Compositions and methods for detecting severe acute respiratory syndrome coronavirus

ABSTRACT

The invention provides compositions and methods for detecting the presence of SARS-coronavirus, for screening anti-SARS coronavirus agents and vaccines, and for reducing infection with plus-strand RNA viruses such as SARS-coronavirus.

This invention was made, in part, with government support under grantnumber N01-A1-25490 awarded by the National Institutes of Health, andgrant number 1N01-AI-25490 awarded by the Emerging Viral Diseases. TheU.S. government has certain rights in the invention.

FIELD OF THE INVENTION

The invention relates to compositions and methods for detecting thepresence of SARS-coronavirus, and for screening anti-SARS coronavirusagents and vaccines. The invention also relates to reducing infectionwith plus-strand RNA viruses such as SARS-coronavirus. These methods maybe used for increasing the safety of cell cultures that are used inscreening clinical samples for respiratory pathogens other thanSARS-coronavirus.

BACKGROUND OF THE INVENTION

An outbreak of severe acute respiratory syndrome (SARS) emerged inGuangdong Province, People's Republic of China in November 2002. FromChina, SARS spread to 30 other countries and as of Aug. 7, 2003, thisoutbreak resulted in 8,422 reported cases, of which 918 were fatal.Through the coordinated efforts of laboratories around the world, anovel coronavirus, SARS-coronavirus (SARS-CoV), was identified as thecausative agent of SARS (Drosten, et al., 2003, N. Engl. J. Med.348:1967-1976; Fouchier, et al., 2003, Nature 423:240; Ksiazek, et al.,2003, N. Engl. J. Med. 348:1953-1966; Peiris, et al., 2003, Lancet361:1319-1325; Poutanen, et al., 2003, N. Engl. J. Med. 348:1995-2005).This discovery was quickly followed by the publication of the completegenomic sequences of two SARS-CoV isolates and identification ofspecific subgenomic RNAs and proteins involved in replication (Marra, etal., 2003, Science 300:1399-1404; Rota, et al., 2003, Science300:1394-1399; Thiel, et al., 2003, J. Gen. Virol. 84:2305-2315).Phylogenetic analysis of the SARS-CoV replicase gene demonstrated thatdespite a number of unique features, SARS-CoV is most closely related togroup 2 coronaviruses, which include mouse hepatitis virus (MHV), bovinecoronavirus (BCoV) and human coronavirus OC43 (HCoV-OC43) (Snijder, etal., 2003, J. Mol. Biol. 331:991-1004).

SARS-CoV has been detected using the Vero E6 cell line and fetal rhesusmonkey kidney cells, which are the only cell lines reported to besusceptible to SARS-CoV. Susceptibility of these cells to SARS-CoV wasbased on observing a cytopathic effect (CPE) post inoculation withSARS-CoV. However, many coronaviruses cause persistent infections incell cultures and some show little evidence of CPE. Thus, using CPE toidentify entry of SARS-CoV or abortive replication is insensitive,misleading, and does not correctly identify virus entry and/orreplication.

SARS-CoV has also been detected using virus titration techniques, RT-PCRspecific to SARS-CoV genomic RNA, and immunofluorescence assay. However,these methods are laborious, and do not distinguish between entry andreplication of the virus.

Thus, there remains a need for compositions and methods for detectingthe presence of SARS-coronavirus, for screening anti-SARS coronavirusagents and vaccines. There is also a need for increasing the safety ofcell cultures that are routinely used in laboratories and that maysupport infection by plus-strand RNA viruses, such as SARS-coronavirus.

SUMMARY OF THE INVENTION

The invention provides compositions and methods for detecting thepresence of SARS-coronavirus, and for screening anti-SARS coronavirusagents and vaccines. Also provided are compositions and methods forreducing infection with plus-strand RNA viruses such asSARS-coronavirus.

In one embodiment, the invention provides a method for detectingreplication of severe acute respiratory syndrome coronavirus(SARS-coronavirus) in a sample, comprising detecting the presenceSARS-coronavirus sgRNA in the sample. In one example, sgRNA comprises atleast a portion of a leader sequence. In another example, the sgRNAcomprises a gene encoding a SARS-coronavirus polypeptide. In anotherembodiment, the method further comprises detecting SARS-coronavirusgRNA. While not intending to limit the method of detection, in oneembodiment, the detecting of gRNA and/or sgRNA is by reversetranscriptase PCR, ribonuclease protection assay, and/or by Northernblot. In another embodiment, the method further comprises quantitatingsgRNA and/or gRNA. In yet a further embodiment, the method furthercomprises detecting one or more SARS-coronavirus polypeptide using, forexample, immunofluorescence and/or Western blot. In an additionalembodiment, the method further comprises detecting SARS-coronavirusparticles.

The invention also provides a method for detecting the presence ofsevere acute respiratory syndrome coronavirus (SARS-coronavirus) in asample, comprising: a) providing: (i) a sample; and (ii) cells chosenfrom HEK-293T, Huh-7, Mv1Lu, pRHMK and pCMK; b) inoculating the cellswith the sample to produce inoculated cells; and c) detecting thepresence of the SARS-coronavirus in the inoculated cells. In oneembodiment, the detecting step comprises detecting the presence of aSARS-coronavirus polypeptide (such as Nucleocapsid (N), SpikeGlycoprotein (S), Matrix (M), E protein, and Replicase proteins) by, forexample, immunofluorescence and/or Western blot. Alternatively, or inaddition, the detecting may comprise detecting the presence ofSARS-coronavirus gRNA and/or sgRNA.

While not intending to limit the type or source of cell in any of theinvention's methods, in one embodiment, the cells comprises a transgeniccell and/or wild type cell. In a preferred embodiment, the transgeniccell comprises Mv1Lu-hF deposited as ATCC accession number PTA-4737.Alternatively, the transgenic cell comprises a cell line establishedfrom a transgenic cell line designated Mv1Lu-hF, wherein the establishedcell line has a property selected from the group consisting of (a)increased sensitivity to at least one virus selected from the groupconsisting of influenza A virus, influenza B virus and parainfluenzavirus 3, as compared to the Mv1Lu cell line, and (b) enhancedproductivity of infectious virions upon inoculation with at least onevirus selected from the group one consisting of influenza A virus,influenza B virus and parainfluenza virus 3, as compared to the Mv1Lucell line. In another embodiment, the transgenic cell comprises atransgenic mink lung epithelial cell line expressing human furin,wherein the cell line has a property selected from the group consistingof (a) increased sensitivity to at least one virus selected from thegroup consisting of influenza A virus, influenza B virus andparainfluenza virus 3, as compared to Mv1Lu, and (b) enhancedproductivity of infectious virions upon inoculation with at least onevirus selected from the group one consisting of influenza A virus,influenza B virus and parainfluenza virus 3, as compared to Mv1Lu. In afurther embodiment, the inoculating step comprises contacting the cells(whether wild type and/or transgenic) with a protease inhibitor.

While not intending to limit the type of culture in any of theinvention's methods, the cells may be in single cell type culture, inmixed cell type culture with a second cell type, and/or are frozen insitu. Also without limiting the source or type of sample in any of theinvention's methods, sample is isolated from a mammal, preferably from ahuman.

The invention further provides a method for detecting the presence ofsevere acute respiratory syndrome coronavirus (SARS-coronavirus) in afirst sample and in a second sample, comprising: a) providing: (i) afirst sample; (ii) a second sample; b) contacting test cells chosen fromHEK-293T, Huh-7, Mv1Lu, pRHMK and pCMK with: (i) the first sample toproduce a first treated sample; and (ii) the second sample to produce asecond treated sample; wherein the exposing is under conditions suchthat the test cells are infected with SARS-coronavirus; c) detecting thepresence of SARS-coronavirus gRNA and SARS-coronavirus sgRNA, whereinthe detecting indicates the presence of the SARS-coronavirus. In oneembodiment, the detecting step comprises detecting one or more of: i)absence of SARS-coronavirus gRNA in the first treated sample; ii)reduced level of SARS-coronavirus sgRNA in the first treated samplecompared to the level of sgRNA in the second treated sample; and iii)reduced ratio of SARS-coronavirus sgRNA level to SARS-coronavirus gRNAlevel in the first treated sample compared to in the second treatedsample; wherein the detecting indicates that the first sample contains areduced level of SARS-coronavirus compared to the second sample. In oneembodiment, the first sample is from a mammal treated with an agentidentified according to any method, and the second sample is from themammal that is not treated with the agent. In another embodiment, thefirst sample is from a mammal treated with a first concentration of anagent identified according to any method, and the second sample is fromthe mammal treated with a second concentration of the agent, wherein thefirst and second concentrations are different. In yet anotherembodiment, the first sample is from a mammal treated with a first agentidentified according to any method, and the second sample is from themammal treated with a second agent identified according to any method,wherein the first and second agent are different.

The invention also provides a method for identifying a test agent asaltering (such as reducing or increasing) replication of severe acuterespiratory syndrome coronavirus (SARS-coronavirus) in a cell,comprising: a) providing cells treated with a first test agent, whereinthe cells are chosen from HEK-293T, Huh-7, Mv1Lu, pRHMK and pCMK; and b)detecting an altered level of replication of cells treated with thefirst test agent compared to a level of replication of the cells nottreated with the first test agent, wherein the detecting identifies thefirst test agent as altering replication of severe acute respiratorysyndrome coronavirus (SARS-coronavirus) in a cell. Without limiting themethod of detection, in one embodiment, the detecting step may comprisedetecting SARS-coronavirus sgRNA, gRNA, polypeptide and/or virusparticle. In another embodiment, the detecting comprises detecting oneor more of: i) absence of SARS-coronavirus gRNA in the treated cells;ii) reduced level of SARS-coronavirus sgRNA in the treated cellscompared to the level of sgRNA in the cells that are not treated withthe first test agent; and iii) reduced ratio of SARS-coronavirus sgRNAlevel relative to SARS-coronavirus gRNA level in the treated cellscompared to in the cells that are not treated with the first test agent;wherein the detecting identifies the first test agent as reducingreplication of severe acute respiratory syndrome coronavirus(SARS-coronavirus) in a cell. Without limiting the use or methodology,it may be desirable to compare the efficacy of two potential drugs, bycomparing their effect on only sgRNA by detecting comprises detectingone or more of: i) reduced level of SARS-coronavirus sgRNA in the cellstreated with a second test agent compared to the level of sgRNA in thecells treated with the first test agent; and ii) reduced ratio ofSARS-coronavirus sgRNA level to SARS-coronavirus gRNA level in the cellstreated with a second test agent compared to the ratio in the cellstreated with the first test agent. In an exemplary embodiment, detectingone or more of: a) an increased reduction in the level ofSARS-coronavirus sgRNA in the cells treated with the first test agentcompared to the cells treated with the second test agent, and b) anincreased reduction in the ratio of SARS-coronavirus sgRNA level toSARS-coronavirus gRNA level in the cells treated with the first testagent compared to the cells treated with the second test agent, whereinthe detecting identifies the first test agent as more efficacious thanthe second test agent in reducing replication of severe acuterespiratory syndrome coronavirus (SARS-coronavirus) in a cell.

Additionally provided herein is a method for reducing replication ofsevere acute respiratory syndrome coronavirus (SARS-coronavirus) in amammal, comprising administering a therapeutic amount of a test agent tothe mammal, wherein the test agent is identified according to the abovemethod.

The invention also provides a method for producing one or more of severeacute respiratory syndrome coronavirus (SARS-coronavirus) particles andSARS-coronavirus polypeptide, comprising: a) providing: (i)SARS-coronavirus; and (ii) a cell type chosen from HEK-293T, Huh-7,Mv1Lu, pRHMK and pCMK; and b) inoculating the cell type with the virusunder conditions such that the inoculated cell produces one or more ofSARS-coronavirus and SARS-coronavirus polypeptide.

Moreover, provided by the invention is an antibody specific for one ormore SARS-coronavirus antigen that is produced by a cell chosen fromHEK-293T, Huh-7, Mv1Lu, pRHMK and pCMK. The antibody may comprises apolyclonal antibody, monoclonal antibody, and/or humanized antibody.

Also provided is a severe acute respiratory syndrome coronavirus(SARS-coronavirus) vaccine produced using cells chosen from HEK-293T,Huh-7, Mv1Lu, pRHMK and pCMK.

The invention additionally provides a method for immunizing a mammalagainst severe acute respiratory syndrome coronavirus(SARS-coronavirus), comprising administering to a mammal a vaccineproduced according any method, wherein the administering generating animmune response in the mammal against severe acute respiratory syndromecoronavirus (SARS-coronavirus).

Also provided herein is a composition comprising (i) cells susceptibleto a virus that is not a plus-strand RNA virus, and (ii) proteaseinhibitor. The plus-strand RNA virus is exemplified by Adenovirus,Arenaviridae, Baculoviridae, Birnaviridae, Bunyaviridae, Caliciviridae,Cardiovirus, Corticoviridae, Cystoviridae, Epstein-Barr virus,Enterovirus, Filoviridae, Foot-and-mouth disease virus, Hepadnviridae,Hepatitis virus, Herpesviridae, Immunodeficiency virus, Influenza virus,Inoviridae, Iridoviridae, Orthomyxoviridae, Papovaviru, Paramyxoviridae,Parvoviridae, Poliovirus, Polydnaviridae, Poxyviridae, Reoviridae,Retrovirus, Rhabdoviridae, Rhinoviridae, Semliki Forest virus,Tetraviridae, Toroviridae, Vaccinia virus, and Vesicular stomatitisvirus.

Further provided herein is a method for detecting a virus that is not aplus-strand RNA virus in a sample, comprising: a) providing: i) asample; ii) cells susceptible to the virus that is not a plus-strand RNAvirus; and iii) one or more protease inhibitor; b) contacting the cellsand the sample in the presence of the protease inhibitor to producecontacted cells, wherein replication of the virus that is not aplus-strand RNA virus in the contacted cells is not reduced relative toreplication of the virus that is not a plus-strand RNA virus in cellsnot contacted with the protease inhibitor, and wherein replication of aplus-strand RNA virus in the cells contacted with the protease inhibitoris reduced relative to replication of the plus-strand RNA virus in cellsnot contacted with the protease inhibitor. In one embodiment, theplus-strand RNA virus is chosen from togavirus, flavivirus, coronavirus,and picornavirus.

The invention also provides a composition comprising (i) cellssusceptible to a virus chosen from influenza virus, parainfluenza virus,adenovirus, and respiratory syncytial virus, and (ii) proteaseinhibitor.

Also provided herein, is method for detecting a virus chosen frominfluenza virus, parainfluenza virus, adenovirus, and respiratorysyncytial virus in a sample, comprising: a) providing: i) a sample; ii)cells susceptible to the virus; and iii) one or more protease inhibitor;b) contacting the cells and the sample in the presence of the proteaseinhibitor to produce contacted cells, wherein replication of thecontacted cells by the virus is not reduced relative to cells notcontacted with the protease inhibitor, and wherein replication of thecontacted cells by severe acute respiratory syndrome coronavirus(SARS-coronavirus) is reduced relative to cells not contacted with theprotease inhibitor. In one embodiment, the influenza virus is chosenfrom influenza A, influenza B, and influenza C, the parainfluenza virusis chosen from parainfluenza 1, parainfluenza 2, and parainfluenza 3.Without limiting the cell type in this or any of the invention'smethods, in one embodiment, the cells comprise a transgenic cell (suchas Mv1Lu-hF) and/or wild type cell. Also without limiting the nature ofthe culture used in this and in any other of the invention's methods,the cells may be in single cell type culture, mixed cell type culture(comprising a wild type cell and/or a transgenic cell), and/or arefrozen in situ. In one embodiment, the inoculated Mv1Lu cells areincubated with the sample for up to 24 hours and/or up to 48 hours.Without limiting the type of sample in any of the invention's methods,the sample is isolated from a mammal that has been treated with an agentthat is suspected of reducing replication of SARS-coronavirus in a cell.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows an exemplary multiplex RT-PCR assay for the detection ofSARS-CoV replication. Amplification of G3PDH, SARS-CoV genomic RNA(gRNA) and subgenomic RNA (sgRNA) from RNA at 1 and 24 hours postinoculation of Vero E6 cells inoculated with serial dilutions ofSARS-CoV. Mock inoculation (M). Negative images are shown. Figure isrepresentative of 3 experiments each performed in duplicate.

FIG. 2 shows susceptibility of monkey kidney cells to SARS-CoV. (A)Amplification of G3PDH, SARS-CoV gRNA and sgRNA at 1, 24, 48 hpost-inoculation (p.i.). African green monkey cells (Vero E6), primaryrhesus monkey kidney cells (pRhMK), primary cynomologous monkey kidneycells (pCMK). Mock inoculated cells (M) and baby hamster kidney cells(BHK21) included as negative controls. Negative images are shown.Results are representative of 2 experiments performed in duplicate. (B)Titration of cell supernatants in Vero E6 cells (TCID₅₀). Graph depictsthe average of two to three experiments each in triplicate.

FIG. 3 shows susceptibility of cells expressing known coronavirusreceptors. Amplification of G3PDH, SARS-CoV gRNA and sgRNA at 1, 24 and48 h p.i. Human lung fibroblasts (MRC-5), canine kidney (MDCK), felinelung epithelia (AK-D), murine fibroblast (L2), and human rectal tumor(HRT-18). Vero E6 included as a positive control, mock infected asnegative control (M). * denotes non-specific amplification product.Negative images are shown. Figure is representative of 2 experimentsperformed in duplicate.

FIG. 4 shows susceptibility of clinically relevant cells to SARS-CoV.(A) Amplification of G3PDH, SARS-CoV gRNA and sgRNA at 1, 24 and 48 hp.i. Mixed monolayer of mink lung cells and human lung cells (R-Mix),Mink lung cells (Mv1Lu), human lung cells (A549) and human embryoniclung cells (HEL). Mock inoculated cells included as negative control.Negative images are shown. Figure is representative of two experimentsperformed in duplicate. (B) Titration of cell supernatants in Vero E6cells (TCID₅₀). Graph is average of 2 experiments performed intriplicate.

FIG. 5 shows susceptibility of human cell lines to SARS-CoV. (A)Amplification of G3PDH, SARS-CoV gRNA and sgRNA at 1, 24 and 48 h p.i.Human embryonic kidney (HEK-293T) and human liver carcinoma cells(Huh-7). Mock inoculated cells were included as a negative control (M).Negative images are shown. This Figure is representative of threeexperiments performed in duplicate.

FIG. 6 shows the effect of human APN on susceptibility of cells. (A)Amplification of G3PDH, SARS-CoV gRNA and sgRNA at 1, 24 and 48 h p.i.Murine epithelia cells (CMT-93), CMT-93 expressing human APN (hAPN)(CMT-93/hAPN), baby hamster kidney cells (BHK-21) and BHK-21 expressinghAPN (BHK-21/hAPN). Mock inoculated cells included as negative control;Huh-7 cells included as positive control. Negative images are shown.Figure is representative of three experiments performed in triplicate.(B) FACS analysis of APN expression. Cells transfected with hAPN aredepicted by the solid line, cells without APN are depicted by the dashedline, staining with isotype control antibody is represented by theshaded curve.

FIG. 7A-I shows the genomic RNA sequence of SARS-CoV Urbani (GenBankaccession # AY278741) (SEQ ID NO:1).

FIG. 8A-J shows the genomic RNA sequence of SARS-CoV Tor2 (GenBankaccession # AY274119) (SEQ ID NO:2).

FIG. 9A-I shows the genomic RNA sequence of SARS-CoV CUHK-W1 (GenBankaccession # AY278554) (SEQ ID NO:3).

FIG. 10 shows a partial genomic RNA sequence of SARS-CoV Shanghai LY(GenBank accession # H012999) (SEQ ID NO:4) orf1a polyprotein gene.

FIG. 11A-C shows a partial genomic RNA sequence of SARS-CoV Shanhgai LY(GenBank accession # H012999) (SEQ ID NO:5) orf1ab polyprotein and orf1apolyprotein genes.

FIG. 12A-B shows a partial genomic RNA sequence of SARS-CoV Shanhgai LY(GenBank accession # H012999) (SEQ ID NO:6) orf1ab polyprotein, Spikeglycoprotein, and Orf3a genes.

FIG. 13 shows a partial genomic RNA sequence of SARS-CoV Shanhgai LY(GenBank accession # H012999) (SEQ ID NO:7) Orf7a, Orf7b, Orf8A, Orf8b,and Nucleocapsid protein genes.

FIG. 14A-B shows a partial genomic RNA sequence of SARS-CoV Shanghai QXC(GenBank accession # AH013000) (SEQ ID NO:8) orf1a polyprotein, andorf1ab polyprotein genes.

FIG. 15A-B shows a partial genomic RNA sequence of SARS-CoV Shanghai QXC(GenBank accession # AH013000) (SEQ ID NO:9) orf1ab polyprotein gene.

FIG. 16 shows a partial genomic RNA sequence of SARS-CoV Shanghai QXC(GenBank accession # AH013000) (SEQ ID NO:10) of the Orf3a, Orf4b,envelope protein E, membrane glycoprotein M, Orf6, and Orf7a genes.

FIG. 17 shows a partial genomic RNA sequence of SARS-CoV Shanhgai LY(GenBank accession # AY322208) (SEQ ID NO:11) of the Orf7a gene (partialcds); and Orf7b, Orf8A, Orf8b, and Nucleocapsid protein genes (completecds).

FIG. 18A-B shows a genomic RNA sequence of SARS-CoV Shanghai QXC(GenBank accession # AY322197) (SEQ ID NO:12) orf1ab polyprotein andorf1a polyprotein genes.

FIG. 19 shows a genomic RNA sequence of SARS-CoV Shanghai QXC (GenBankaccession # AY322199) (SEQ ID NO:13) Orf3a gene (partial cds), Orf4b,envelope protein E, membrane glycoprotein M, and Orf6 genes (completecds), and Orf7a gene (partial cds).

FIG. 20 shows a genomic RNA sequence of SARS-CoV Shanghai LY (GenBankaccession # AY322205) (SEQ ID NO:14) orf1ab polyprotein and orf1apolyprotein genes (partial cds).

FIG. 21A-D shows a genomic RNA sequence of SARS-CoV Shanghai LY (GenBankaccession # AY322206) (SEQ ID NO:15) orf1a polyprotein and orf1abpolyprotein genes (partial cds).

FIG. 22 shows a genomic RNA sequence of SARS-CoV ZJ-HZ01 (GenBankaccession # AY322206) (SEQ ID NO:16) Nucleocapsid protein,uncharacterized protein 9b, and uncharacterized protein 9c genes,(complete cds).

FIG. 23 shows the amino acid sequence (SEQ ID NO:17) of Nucleocapsidprotein of SARS-CoV (Urbani) (Genbank Accession Number AY278741).

FIG. 24 shows the amino acid sequence (SEQ ID NO:18) of Nucleocapsidprotein of SARS-CoV (Tor2) (Genbank Accession Number AY274119).

FIG. 25 shows the amino acid sequence (SEQ ID NO:19) of Nucleocapsidprotein of SARS-CoV (Shanghai LY) (A) for Genbank Accession NumberAY322205, and (B) for Genbank Accession Number AY322208.

FIG. 26 shows the amino acid sequence (SEQ ID NO:20) of Nucleocapsidprotein of SARS-CoV (ZJ-HZ01) (Genbank Accession Number AY290752).

FIG. 27 shows the amino acid sequence (SEQ ID NO:21) of the Spikeglycoprotein of SARS-CoV (Urbani) (Genbank Accession Number AY278741).

FIG. 28 shows the amino acid sequence (SEQ ID NO:22) of the Spikeglycoprotein of SARS-CoV (Tor2) (Genbank Accession Number AY274119).

FIG. 29 shows the amino acid sequence (SEQ ID NO:23) of the Spikeglycoprotein of SARS-CoV (Shanghai LY) (Genbank Accession NumberAY322205).

FIG. 30 shows the amino acid sequence (SEQ ID NO:24) of the Matrixprotein of SARS-CoV (A) Urbani (Genbank Accession Number AY278741), (B)Tor2 (Genbank Accession Number AY274119), (C and D) Shanghai QXC(Genbank Accession Numbers AY322199 and AH013000, respectively).

FIG. 31 shows the amino acid sequence (SEQ ID NO:25) of the E protein ofSARS-CoV (A) Urbani (Genbank Accession Number AY278741), (B) Tor2(Genbank Accession Number AY274119), (C and D) Shanghai QXC (GenbankAccession Numbers AY322199 and AH013000, respectively), and (E) UHK-W1(Genbank Accession Number AY278554).

FIG. 32A-C shows the amino acid sequence (SEQ ID NO:26) of thepolyprotein 1a of SARS-CoV Urbani (Genbank Accession Number AY278741).

FIG. 33A-E shows the amino acid sequence (SEQ ID NO:27) of thepolyprotein lab of SARS-CoV (Tor2) (Genbank Accession Number AY274119).

FIG. 34A-B shows the amino acid sequence (SEQ ID NO:28) of thepolyprotein 1b of SARS-CoV (Urbani) (Genbank Accession Number AY278741).

FIG. 35A-E shows the amino acid sequence (SEQ ID NO:29) of thepolyprotein 1ab of SARS-CoV (CUHK-W1) (Genbank Accession NumberAY278554).

FIG. 36 shows the amino acid sequence (SEQ ID NO:30) of the polyprotein1a of SARS-CoV (Shanghai QXC) (Genbank Accession Number AY322197).

FIG. 37 shows the amino acid sequence (SEQ ID NO:31) of the polyprotein1ab of SARS-CoV (Shanghai QXC) (Genbank Accession Number AY322197).

FIG. 38 shows the amino acid sequence (SEQ ID NO:32) of the polyprotein1a of SARS-CoV (Shanghai LY) (Genbank Accession Number AY322197).

FIG. 39 shows the amino acid sequence (SEQ ID NO:33) of the polyprotein1ab of SARS-CoV (Shanghai LY) (Genbank Accession Number AY322197).

FIG. 40 shows the amino acid sequence (SEQ ID NO:34) of Nucleocapsidprotein of SARS-CoV (CUHK-W1) (Genbank Accession Number AY278554).

FIG. 41 shows the amino acid sequence (SEQ ID NO:35) of Spike protein ofSARS-CoV (CUHK-W1) (Genbank Accession Number AY278554).

DEFINITIONS

To facilitate understanding of the invention, a number of terms aredefined below.

As used herein, the term “cell type,” refers to any cell, regardless ofits source or characteristics. A cell type includes a “wild-type cell”(i.e., a cell whose genome has not been manipulated by man), and a“transgenic cell.”

As used herein, the term “microorganism” refers to any organism ofmicroscopic or ultramicroscopic size including, but not limited to,viruses, bacteria, and protozoa.

As used herein, the term “culture” refers to a composition, whetherliquid, gel, or solid, which contains one or more microorganisms and/orone or more cells. A culture of organisms and/or cells can be pure ormixed. For example, the terms “pure culture” of a microorganism as usedherein refers to a culture in which the microorganisms present are ofonly one strain of a single species of a particular genus. This is incontrast to a “mixed culture” of microorganisms which refers to aculture in which more than one strain of a single genus and/or speciesof microorganism is present.

As used herein, the terms “culture media,” and “cell culture media,”refer to media that are suitable to support maintenance and/or growth ofcells in vitro (i.e., cell cultures).

A “primary cell” is a cell which is directly obtained from a tissue ororgan of an animal whether or not the cell is in culture.

A “cultured cell” is a cell which has been maintained and/or propagatedin vitro. Cultured cells include primary cultured cells and cell lines.

“Primary cultured cells” are primary cells which are in in vitro cultureand which preferably, though not necessarily, are capable of undergoingten or fewer passages in in vitro culture before senescence and/orcessation of proliferation.

The terms “cell line” and “immortalized cell” refer to a cell which iscapable of a greater number of cell divisions in vitro before cessationof proliferation and/or senescence as compared to a primary cell fromthe same source. A cell line includes, but does not require, that thecells be capable of an infinite number of cell divisions in culture. Thenumber of cell divisions may be determined by the number of times a cellpopulation may be passaged (i.e., subcultured) in in vitro culture.Passaging of cells is accomplished by methods known in the art. Briefly,a confluent or subconfluent population of cells which is adhered to asolid substrate (e.g., plastic Petri dish) is released from thesubstrate (e.g., by enzymatic digestion), and a proportion (e.g., 10%)of the released cells is seeded onto a fresh substrate. The cells areallowed to adhere to the substrate, and to proliferate in the presenceof appropriate culture medium. The ability of adhered cells toproliferate may be determined visually by observing increased coverageof the solid substrate over a period of time by the adhered cells.Alternatively, proliferation of adhered cells may be determined bymaintaining the initially adhered cells on the solid support over aperiod of time, removing and counting the adhered cells and observing anincrease in the number of maintained adhered cells as compared to thenumber of initially adhered cells.

Cell lines may be generated spontaneously or by transformation. A“spontaneous cell line” is a cell line which arises during routineculture of cells. A “transformed cell line” refers to a cell line whichis generated by the introduction of a “transgene” comprising nucleicacid (usually DNA) into a primary cell or into a finite cell line by wayof human intervention.

Cell lines include, but are not limited to, finite cell lines andcontinuous cell lines. As used herein, the term “finite cell line”refers to a cell line which is capable of a limited number (from about 1to about 50, more preferably from about 1 to about 40, and mostpreferably from about 1 to about 20) of cell divisions prior tosenescence.

The term “continuous cell line” refers to a cell line which is capableof more than about 50 (and more preferably, an infinite number of) celldivisions. A continuous cell line generally, although not necessarily,also has the general characteristics of a reduced cell size, highergrowth rate, higher cloning efficiency, increased tumorigenicity, and/ora variable chromosomal complement as compared to the finite cell line orprimary cultured cells from which it is derived.

The term “transgene” as used herein refers to any nucleic acid sequencewhich is introduced into the cell by experimental manipulations. Atransgene may be an “endogenous DNA sequence” or a “heterologous DNAsequence” (i.e., “foreign DNA”). The term “endogenous DNA sequence”refers to a nucleotide sequence which is naturally found in the cellinto which it is introduced so long as it does not contain somemodification (e.g., a point mutation, the presence of a selectablemarker gene, etc.) relative to the naturally-occurring sequence. Theterm “heterologous DNA sequence” refers to a nucleotide sequence whichis ligated to, or is manipulated to become ligated to, a nucleic acidsequence to which it is not ligated in nature, or to which it is ligatedat a different location in nature. Heterologous DNA is not endogenous tothe cell into which it is introduced, but has been obtained from anothercell. Heterologous DNA also includes an endogenous DNA sequence whichcontains some modification. Generally, although not necessarily,heterologous DNA encodes RNA and proteins that are not normally producedby the cell into which it is expressed. Examples of heterologous DNAinclude reporter genes, transcriptional and translational regulatorysequences, selectable marker proteins (e.g., proteins which confer drugresistance), etc.

The term “nucleotide sequence of interest” refers to any nucleotidesequence, the manipulation of which may be deemed desirable for anyreason, by one of ordinary skill in the art. Nucleotide sequences ofinterest include, but are not limited to, coding sequences of structuralgenes (e.g., reporter genes, selection marker genes, oncogenes, drugresistance genes, growth factors, etc.), and non-coding regulatorysequences which do not encode an mRNA or protein product, (e.g.,promoter sequence, polyadenylation sequence, termination sequence,enhancer sequence, etc.).

As used herein, the singular forms “a,” “an” and “the” include bothsingular and plural references unless the content clearly dictatesotherwise.

As used herein, the term “or” when used in the expression “A or B,”where A and B refer to a composition, disease, product, etc., means one,or the other, or both.

The terms “chosen from A, B and C” and “chosen from one or more of A, Band C” are equivalent terms that mean selecting any one of A, B, and C,or any combination of A, B, and C.

As used herein, the term “comprising” when placed before the recitationof steps in a method means that the method encompasses one or more stepsthat are additional to those expressly recited, and that the additionalone or more steps may be performed before, between, and/or after therecited steps. For example, a method comprising steps a, b, and cencompasses a method of steps a, b, x, and c, a method of steps a, b, c,and x, as well as a method of steps x, a, b, and c. Furthermore, theterm “comprising” when placed before the recitation of steps in a methoddoes not (although it may) require sequential performance of the listedsteps, unless the content clearly dictates otherwise. For example, amethod comprising steps a, b, and c encompasses, for example, a methodof performing steps in the order of steps a, c, and b, the order ofsteps c, b, and a, and the order of steps c, a, and b, etc.

Unless otherwise indicated, all numbers expressing quantities ofingredients, properties such as molecular weight, reaction conditions,and so forth as used herein, are to be understood as being modified inall instances by the term “about.” Accordingly, unless indicated to thecontrary, the numerical parameters herein are approximations that mayvary depending upon the desired properties sought to be obtained by thepresent invention. At the very least, and without limiting theapplication of the doctrine of equivalents to the scope of the claims,each numerical parameter should at least be construed in light of thenumber of reported significant digits and by applying ordinary roundingtechniques. Notwithstanding that the numerical ranges and parametersdescribing the broad scope of the invention are approximations, thenumerical values in the specific examples are reported as precisely aspossible. Any numerical value, however, inherently contains standarddeviations that necessarily result from the errors found in thenumerical value's testing measurements.

The term “not” when preceding, and made in reference to, anyparticularly named molecule (e.g., nucleic acid sequence such as “gRNA,”“sgRNA,” amino acid sequence such as “Nucleocapsid,” “Spike,” “Matrix,”“E protein,” and “Replicase proteins,” etc.), and/or phenomenon (e.g.,susceptibility, permissivity, infection with a microorganism, binding toa molecule, expression of a nucleic acid sequence, transcription of anucleic acid sequence, enzyme activity, etc.) means that only theparticularly named molecule or phenomenon is excluded.

The term “altering” and grammatical equivalents as used herein inreference to the level of any molecule (e.g., nucleic acid sequence suchas “gRNA,” “sgRNA,” amino acid sequence such as “Nucleocapsid,” “Spike,”“Matrix,” “E protein,” and “Replicase proteins,” etc.), and/orphenomenon (e.g., susceptibility, permissivity, infection with amicroorganism, binding to a molecule, expression of a nucleic acidsequence, transcription of a nucleic acid sequence, enzyme activity,etc.) refers to an increase and/or decrease in the quantity of themolecule and/or phenomenon, regardless of whether the quantity isdetermined objectively, and/or subjectively. For example, “alteringreplication” of a virus includes increasing and/or decreasing thequantity of any one or more of the steps of adsorption (e.g., receptorbinding) to a cell, entry into a cell (such as by endocytosis),introducing the viral genome sequence into the cell, uncoating the viralgenome, initiating transcription of genomic RNA, producing subgenomicRNA, directing expression of SARS-CoV encapsidation proteins,encapsidating the replicated viral nucleic acid sequence with theencapsidation proteins into a viral particle, release of theencapsidated virus from the cell, and infection of other cells by thereleased virus.

Unless defined otherwise in reference to the level of molecules and/orphenomena, the terms “increase,” “elevate,” “raise,” and grammaticalequivalents when in reference to the level of any molecule (e.g.,nucleic acid sequence such as “gRNA,” “sgRNA,” amino acid sequence suchas “Nucleocapsid,” “Spike,” “Matrix,” “E protein,” and “Replicaseproteins,” etc.), and/or phenomenon (e.g., susceptibility, permissivity,infection with a microorganism, binding to a molecule, expression of anucleic acid sequence, transcription of a nucleic acid sequence, enzymeactivity, etc.) in a first sample relative to a second sample, mean thatthe quantity of the molecule and/or phenomenon in the first sample ishigher than in the second sample by any amount that is statisticallysignificant using any art-accepted statistical method of analysis. Inone embodiment, the increase may be determined subjectively, for examplewhen a patient refers to their subjective perception of diseasesymptoms, such as pain, difficulty in breathing, clarity of vision,nausea, tiredness, etc. In another embodiment, the quantity of themolecule and/or phenomenon in the first sample is at least 10% greaterthan, at least 25% greater than, at least 50% greater than, at least 75%greater than, and/or at least 90% greater than the quantity of the samemolecule and/or phenomenon in a second sample.

Unless defined otherwise in reference to the level of molecules and/orphenomena, the terms “reduce,” “inhibit,” “diminish,” “suppress,”“decrease,” and grammatical equivalents when in reference to the levelof any molecule (e.g., nucleic acid sequence such as “gRNA,” “sgRNA,”amino acid sequence such as “Nucleocapsid,” “Spike,” “Matrix,” “Eprotein,” and “Replicase proteins,” etc.), and/or phenomenon (e.g.,susceptibility, permissivity, infection with a microorganism, binding toa molecule, expression of a nucleic acid sequence, transcription of anucleic acid sequence, enzyme activity, etc.) in a first sample relativeto a second sample, mean that the quantity of molecule and/or phenomenonin the first sample is lower than in the second sample by any amountthat is statistically significant using any art-accepted statisticalmethod of analysis. In one embodiment, the reduction may be determinedsubjectively, for example when a patient refers to their subjectiveperception of disease symptoms, such as pain, difficulty in breathing,clarity of vision, nausea, tiredness, etc. In another embodiment, thequantity of molecule and/or phenomenon in the first sample is at least10% lower than, at least 25% lower than, at least 50% lower than, atleast 75% lower than, and/or at least 90% lower than the quantity of thesame molecule and/or phenomenon in a second sample.

Reference herein to any specifically named protein (such as“Nucleocapsid,” “Spike,” “Matrix,” “E protein,” and “Replicaseproteins,” etc.) refers to any and all equivalent fragments, fusionproteins, and variants of the specifically named protein, having atleast one of the biological activities (such as those disclosed hereinand/or known in the art) of the specifically named protein, wherein thebiological activity is detectable by any method.

The term “fragment” when in reference to a protein (such as“Nucleocapsid,” “Spike,” “Matrix,” “E protein,” and “Replicaseproteins,” etc.) refers to a portion of that protein that may range insize from four (4) contiguous amino acid residues to the entire aminoacid sequence minus one amino acid residue. Thus, a polypeptide sequencecomprising “at least a portion of an amino acid sequence” comprises fromfour (4) contiguous amino acid residues of the amino acid sequence tothe entire amino acid sequence.

The term “fusion protein” refers to two or more polypeptides that areoperably linked. The term “operably linked” when in reference to therelationship between nucleic acid sequences and/or amino acid sequencesrefers to linking the sequences such that they perform their intendedfunction. For example, operably linking a promoter sequence to anucleotide sequence of interest refers to linking the promoter sequenceand the nucleotide sequence of interest in a manner such that thepromoter sequence is capable of directing the transcription of thenucleotide sequence of interest and/or the synthesis of a polypeptideencoded by the nucleotide sequence of interest. The term also refers tothe linkage of amino acid sequences in such a manner so that afunctional protein is produced.

The term “variant” of a protein (such as “Nucleocapsid,” “Spike,”“Matrix,” “E protein,” and “Replicase proteins,” etc.) as used herein isdefined as an amino acid sequence which differs by insertion, deletion,and/or conservative substitution of one or more amino acids from theprotein of which it is a variant. The term “conservative substitution”of an amino acid refers to the replacement of that amino acid withanother amino acid which has a similar hydrophobicity, polarity, and/orstructure. For example, the following aliphatic amino acids with neutralside chains may be conservatively substituted one for the other:glycine, alanine, valine, leucine, isoleucine, serine, and threonine.Aromatic amino acids with neutral side chains which may beconservatively substituted one for the other include phenylalanine,tyrosine, and tryptophan. Cysteine and methionine are sulphur-containingamino acids which may be conservatively substituted one for the other.Also, asparagine may be conservatively substituted for glutamine, andvice versa, since both amino acids are amides of dicarboxylic aminoacids. In addition, aspartic acid (aspartate) my be conservativelysubstituted for glutamic acid (glutamate) as both are acidic, charged(hydrophilic) amino acids. Also, lysine, arginine, and histidine my beconservatively substituted one for the other since each is a basic,charged (hydrophilic) amino acid. Guidance in determining which and howmany amino acid residues may be substituted, inserted or deleted withoutabolishing biological and/or immunological activity may be found usingcomputer programs well known in the art, for example, DNAStar™ software.In one embodiment, the sequence of the variant has at least 95%identity, at least 90% identity, at least 85% identity, at least 80%identity, at least 75% identity, at least 70% identity, and/or at least65% identity with the sequence of the protein in issue.

Reference herein to any specifically named nucleotide sequence (such asa sequence encoding “Nucleocapsid,” “Spike,” “Matrix,” “E protein,” and“Replicase proteins,” etc.) includes within its scope any and allequivalent fragments, homologs, and sequences that hybridize underhighly stringent and/or medium stringent conditions to the specificallynamed nucleotide sequence, and that have at least one of the biologicalactivities (such as those disclosed herein and/or known in the art) ofthe specifically named nucleotide sequence, wherein the biologicalactivity is detectable by any method.

The “fragment” or “portion” may range in size from an exemplary 5, 10,20, 50, or 100 contiguous nucleotide residues to the entire nucleic acidsequence minus one nucleic acid residue. Thus, a nucleic acid sequencecomprising “at least a portion of” a nucleotide sequence comprises fromfive (5) contiguous nucleotide residues of the nucleotide sequence tothe entire nucleotide sequence.

The term “homolog” of a specifically named nucleotide sequence refers toan oligonucleotide sequence which exhibits greater than 50% identity tothe specifically named nucleotide sequence. Alternatively, or inaddition, a homolog of a specifically named nucleotide sequence isdefined as an oligonucleotide sequence which has at least 95% identity,at least 90% identity, at least 85% identity, at least 80% identity, atleast 75% identity, at least 70% identity, and/or at least 65% identityto nucleotide sequence in issue.

With respect to sequences that hybridize under stringent conditions tothe specifically named nucleotide sequence, high stringency conditionscomprise conditions equivalent to binding or hybridization at 68° C. ina solution containing 5× SSPE, 1% SDS, 5× Denhardt's reagent and 100μg/ml denatured salmon sperm DNA followed by washing in a solutioncontaining 0.1× SSPE, and 0.1% SDS at 68° C. “Medium stringencyconditions” when used in reference to nucleic acid hybridizationcomprise conditions equivalent to binding or hybridization at 42° C. ina solution of 5× SSPE (43.8 g/l NaCl, 6.9 g/l NaH2PO4-H2O and 1.85 g/lEDTA, pH adjusted to 7.4 with NaOH), 0.5% SDS, 5× Denhardt's reagent and100 μg/ml denatured salmon sperm DNA followed by washing in a solutioncomprising 1.0× SSPE, 1.0% SDS at 42° C.

The term “equivalent” when made in reference to a hybridizationcondition as it relates to a hybridization condition of interest meansthat the hybridization condition and the hybridization condition ofinterest result in hybridization of nucleic acid sequences which havethe same range of percent (%) homology. For example, if a hybridizationcondition of interest results in hybridization of a first nucleic acidsequence with other nucleic acid sequences that have from 85% to 95%homology to the first nucleic acid sequence, then another hybridizationcondition is said to be equivalent to the hybridization condition ofinterest if this other hybridization condition also results inhybridization of the first nucleic acid sequence with the other nucleicacid sequences that have from 85% to 95% homology to the first nucleicacid sequence.

As will be understood by those of skill in the art, it may beadvantageous to produce a nucleotide sequence encoding a protein ofinterest, wherein the nucleotide sequence possesses non-naturallyoccurring codons. Therefore, in some embodiments, codons preferred by aparticular prokaryotic or eukaryotic host (Murray et al., Nucl. AcidsRes., 17 (1989)) are selected, for example, to increase the rate ofexpression or to produce recombinant RNA transcripts having desirableproperties, such as a longer half-life, than transcripts produced fromnaturally occurring sequence. The term “naturally occurring” as usedherein when applied to an object (such as cell, tissue, etc.) and/ormolecule (such as amino acid, amino acid sequence, nucleic acid, nucleicacid sequence, codon, etc.) means that the object and/or molecule can befound in nature. For example, a naturally occurring polypeptide sequencerefers to a polypeptide sequence that is present in an organism(including viruses) that can be isolated from a source in nature,wherein the polypeptide sequence has not been intentionally modified byman in the laboratory.

A “composition” comprising a particular polynucleotide sequence and/orcomprising a particular protein sequence as used herein refers broadlyto any composition containing the recited polynucleotide sequence(and/or its equivalent fragments, homologs, and sequences that hybridizeunder highly stringent and/or medium stringent conditions to thespecifically named nucleotide sequence) and/or the recited proteinsequence (and/or its equivalent fragments, fusion proteins, andvariants), respectively. The composition may comprise an aqueoussolution containing, for example, salts (e.g., NaCl), detergents (e.g.,SDS), and other components (e.g., Denhardt's solution, dry milk, salmonsperm DNA, etc.).

The terms nucleotide sequence “comprising a particular nucleic acidsequence” and protein “comprising a particular amino acid sequence” andequivalents of these terms, refer to any nucleotide sequence of interestand to any protein of interest, respectively, that contain theparticularly named nucleic acid sequence (and/or its equivalentfragments, homologs, and sequences that hybridize under highly stringentand/or medium stringent conditions to the specifically named nucleotidesequence) and the particularly named amino acid sequence (and/or itsequivalent fragments, fusion proteins, and variants), respectively. Theinvention does not limit the source (e.g., cell type, tissue, animal,etc.), nature (e.g., synthetic, recombinant, purified from cell extract,etc.), and/or sequence of the nucleotide sequence of interest and/orprotein of interest. In one embodiment, the nucleotide sequence ofinterest and protein of interest include coding sequences of structuralgenes (e.g., probe genes, reporter genes, selection marker genes,oncogenes, drug resistance genes, growth factors, etc.).

DESCRIPTION OF THE INVENTION

The invention provides compositions and methods for detecting thepresence of SARS-coronavirus, and screening anti-SARS-coronavirus drugsand vaccines. These compositions and methods were premised, at least inpart, on the inventors' discovery of a sensitive assay for determiningsusceptibility and/or permissivity of cells to SARS-CoV. The invention'scompositions and methods are useful for culturing SARS-CoV isolates,producing SARS-CoV virions and/or antigens that may be used in vaccineformulations, as antigen preparations for diagnostic applications, andfor screening antiviral drugs. Additional uses of the invention'scompositions and methods may be found in the elucidation of potentialanimal models and the identification of the SARS-CoV receptor(s).

Also provided are compositions and methods for reducing infection withplus-strand RNA viruses such as SARS-coronavirus. In one embodiment, theinvention provides compositions and methods for reducing infection withSARS-coronavirus, without substantially reducing infection with otherrespiratory viruses. These methods are premised, at least in part, onthe inventors' discovery that protease inhibitors do not substantiallyreduce infection of cells by the exemplary respiratory virusesinfluenza, parainfluenza, RSV, and adenovirus (Example 8). This is incontrast to the inhibition in replication of coronaviruses by theprotease inhibitor E64 (Example 8) and the cysteine proteinase inhibitor(2S,3S)transepoxysuccinyl-L-leucylamido-3-methylbutane ethyl ester(Yount et al. PNAS 100:12995-13000 (2003). These methods are useful,where it is desirable to reduce exposure of personnel toSARS-coronavirus in clinical virology laboratories that routinely screenclinical specimen for respiratory pathogens (such as influenza,parainfluenza, RSV, and adenovirus) other than SARS-coronavirus.

In one embodiment, the invention's methods for detectingSARS-coronavirus are exemplified by a multiplex RT-PCR assay fordetecting G3PDH, SARS-CoV genomic RNA (gRNA) and subgenomic RNA (sgRNA).In one embodiment, subgenomic RNA is indicative of virus entry andreplication. The sensitivity of the PCR assay was determined byinoculation of Vero E6 cells with serial dilutions of SARS-CoV. Human,murine, canine, hamster, feline, mink and monkey cells were analyzed atvarious times post-inoculation and supernatants were titered todetermine if cells produced infectious virus.

The invention is further premised on the discovery, using the exemplarymultiplex RT-PCR assay, of mink, human, and monkey cells that arepermissive to SARS-CoV infection. In particular, kidney cells derivedfrom different species of monkey (primary Rhesus monkey kidney cells(pRhMK) and primary Cynomolgous monkey kidney cells (pCMK) werediscovered by the inventors to be susceptible to, and to be productivelyinfected by SARS-CoV. Data herein also shows that mink lung (Mv1Lu)epithelial cells are also susceptible to, and productively infected bySARS-CoV. In addition, the data shows that SARS-CoV does not use thereceptor for serogroup 1 coronaviruses (APN/CD13) or the receptor formurine coronavirus (CEACAM 1a).

The invention is further described under (A) Coronaviruses, (B) SevereAcute Respiratory Syndrome Coronavirus (SARS-CoV), (C) CytophathicEffect Does Not Always Correlate With SARS-CoV infection, (D) SARS-CoVDoes Not Bind to The Group 1 Coronavirus Receptor aminopeptidase N(APN/CD13) and The Group 2 Coronavirus Receptor carcinoembryonic antigen(CEACAM1a), (E) Cells Permissive To SARS-CoV, (F) Exemplary Assays ForDetecting Replication Of SARS-CoV, (G) Detecting Replication of SARS-CoVUsing The Invention's Exemplary Cells, (H) Screening Anti-SARS-CoVAgents, (I) Administering Anti-SARS-CoV Agents, (J) Producing SARS-CoVAnd SARS-CoV polypeptides, and (K) Compositions And Methods For UsingProtease Inhibitors To Reduce SARS-CoV Infection.

A. Coronaviruses

Coronaviruses (order Nidovirales, family Coronaviridae) are a diversegroup of enveloped, positive-stranded RNA viruses. The coronavirusgenome, approximately 27-32 Kb in length, is the largest found in any ofthe RNA viruses. Large Spike (S) glycoproteins protrude from the virusparticle giving coronaviruses a distinctive corona-like appearance whenvisualized by electron microscopy. Coronaviruses infect a wide varietyof species, including canine, feline, porcine, murine, bovine, avian andhuman (Holmes, et al., 1996. Coronaviridae: the viruses and theirreplication, p. 1075-1094. In D. M. K. a. P. M. H. B. N. Fields (ed.),Fields Virology. Lippincott-Raven, Philadelphia, Pa.). However, thenatural host range of each coronavirus strain is narrow, typicallyconsisting of a single species.

Coronaviruses typically bind to target cells through Spike-receptorinteractions and enter cells by receptor mediated endocytosis or fusionwith the plasma membrane (Holmes, et al., 1996, supra). TheSpike-receptor interaction is a strong determinant of speciesspecificity as demonstrated for both group 1 and group 2 coronaviruses.The receptor for group 1 coronaviruses, including human coronavirus 229E(HCoV-229E), feline coronavirus (FCoV) and porcine coronavirus (PCoV)has been identified as aminopeptidase N (APN/CD13) (Delmas, et al.,1992, Nature 357:417-420; Tresnan, et al., 1996, J. Virol. 70:8669-8674;Yeager, et al., 1992, Nature 357:420-422). APN/CD13 is a 150- to 160-kDatype II protein that is a membrane peptidase (Look, et al., 1989, J.Clin. Invest 83:1299-1307). Expression of cDNAs encoding APN in cellsfrom species normally resistant to infection, renders them susceptibleto infection (Delmas, et al., 1992, Nature 357:417-420; Yeager, et al.,1992, Nature 357:420-422). APN is typically used in a species specificmanner (eg. PCoV binds porcine APN, HCoV-229E binds hAPN, etc.)(Benbacer, et al., J. Virol. 71:734-737; Kolb, et al., 1997. J. Gen.Virol. 78 (Pt 11):2795-2802; Wentworth, et al., 2001, J. Virol.75:9741-9752). However, feline APN acts as a universal receptor forgroup 1 coronaviruses (Tresnan, et al., 1996, J. Virol. 70:8669-8674).

The receptor used by MHV, a group 2 coronavirus, was identified as abiliary glycoprotein in the carcinoembryonic antigen (CEA) family of theimmunoglobulin superfamily (CEACAM) (Williams, et al., 1991, Proc. Natl.Acad. Sci. U.S.A 88:5533-5536; Williams, et al., 1990, J. Virol.64:3817-3823). MHV binds a mouse-specific epitope of CEACAM known asCEACAM1a, and it is this species specificity of virus binding that isbelieved to be a principal determinant of the restricted host range ofMHV infection (Compton, et al., 1982, J. Virol. 66:7420-7428). CEACAM1acDNA transfected into MHV resistant cell lines renders the cellssusceptible to infection with MHV-A59 and MHV-JHM (Dveksler, et al.,1996, J. Virol. 70:4142-4145; Dveksler, et al., 1991, J. Virol.65:6881-6891). Additionally, SL/J mice which express an allelic variantof CEACAM1a are resistant to MHV-A59 (Dveksler, et al., 1995, J. Virol.69:543-546).

Upon entry into susceptible cells, the open reading frame (ORF) nearestthe 5′ terminus of the coronavirus genome is translated into a largepolyprotein. This polyprotein is autocatalytically cleaved byviral-encoded proteases, to yield multiple proteins that together serveas a virus-specific, RNA-dependent RNA polymerase (RdRP). The RdRPreplicates the viral genome and generates 3′ coterminal nestedsubgenomic RNAs. Subgenomic RNAs include capped, polyadenylated RNAsthat serve as mRNAs, and antisense subgenomic RNAs complementary tomRNAs. In one embodiment, each of the subgenomic RNA molecules sharesthe same short leader sequence fused to the body of each gene atconserved sequence elements known as intergenic sequences (IGS),transcriptional regulating sequences (TRS) or transcription activationsequences. It has been controversial as to whether the nested subgenomicRNAs are generated during positive or negative strand synthesis;however, recent work favors the model of discontinuous transcriptionduring minus strand synthesis (Sawicki, et al., 1995, Adv. Exp. Med.Biol. 380:499-506; Sawicki and Sawicki Adv. Expt. Biol. 1998, 440:215).

B. Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV)

The terms “SARS coronavirus,” “SARS-CoV,” and “severe acute respiratorysyndrome coronavirus” are equivalent, and are used to refer to an RNAvirus that is the causative agent of severe acute respiratory syndrome(Drosten, et al., 2003, supra; Fouchier, et al., 2003, supra; Ksiazek,et al., 2003, supra; Peiris, et al., 2003, supra; Poutanen, et al.,2003, supra). Exemplary strains of SARS coronavirus include, but are notlimited to, Urbani, Tor2, CUHK-W1, Shanhgai LY, Shanghai QXC, ZJ-HZ01,TW1, HSR 1, WHU, TWY, TWS, TWK, TWJ, TWH, HKU-39849, FRA, TWC3, TWC2,TWC, ZMY 1, BJ03, ZJ01, CUHK-Su10, GZ50, SZ16, SZ3, CUHK-W1, BJ04, AS,Sin2774, GD01, Sin2500, Sin2677, Sin2679, Sin2748, ZJ-HZ01, and BJ01.While the invention is illustrated using SARS-CoV from humans, the term“SARS coronavirus” expressly includes within its scope equivalentcoronaviruses that cause equivalent severe acute respiratory syndromesin other mammals (such as, without limitation, monkey, hamster, mink,ferret, pig, cat, and rabbit), insects (such as mosquito), etc.

The genome of SARS-CoV contains a single stranded (+)-sense RNA.Complete and partial genome sequences of several SARS coronavirusisolates have been reported, including SARS coronavirus Urbani (GenBankaccession # AY278741, FIG. 7), SARS coronavirus Tor2 (GenBank accession# AY274119, FIG. 8), SARS coronavirus CUHK-W1 (GenBank accession #AY278554, FIG. 9), SARS-CoV Shanhgai LY (GenBank accession # H012999,FIGS. 10-13; GenBank accession # AY322205, FIG. 20; GenBank accession #AY322206, FIG. 21), SARS-CoV Shanghai QXC (GenBank accession # AH013000,FIGS. 14-16; GenBank accession # AY322208, FIG. 17; GenBank accession #AY322197, FIG. 18; GenBank accession # AY322199, FIG. 19), and SARS-CoVZJ-HZ01 (GenBank accession # AY322206, FIG. 22),gi¦31416292¦gb¦AY278487.3¦ SARS coronavirusBJ02,gi¦30248028¦gb¦AY274119.3¦ SARS coronavirus TOR2,gi¦30698326¦gb¦AY291451.1¦ SARS coronavirus TW1,gi¦33115118¦gb¦AY323977.2¦SARS coronavirus HSR 1,gi¦35396382¦gb¦AY394850.1¦ SARS coronavirus WHU,gi¦33411459¦dbj¦AP006561.1¦ SARS coronavirus TWY,gi¦33411444¦dbj¦AP006560.1¦ SARS coronavirus TWS,gi¦33411429¦dbj¦AP006559.1¦ SARS coronavirus TWK,gi¦33411414¦dbj¦AP006558.1¦ SARS coronavirus TWJ,gi¦33411399¦dbj¦AP006557.1¦ SARS coronavirus TWH,gi¦30023963¦gb¦AY278491.2¦ SARS coronavirus HKU-39849,gi¦33578015¦gb¦AY310120.1¦ SARS coronavirus FRA,gi¦33518725¦gb¦AY362699.1¦ SARS coronavirus TWC3,gi¦33518724¦gb¦AY362698.1¦ SARS coronavirus TWC2,gi¦30027617¦gb¦AY278741.1¦ SARS coronavirus Urbani,gi¦31873092¦gb¦AY321118.1¦SARS coronavirus TWC,gi¦33304219¦gb¦AY351680.1¦ SARS coronavirus ZMY 1,gi¦31416305¦gb¦AY278490.3¦ SARS coronavirus BJ03,gi¦30910859¦gb¦AY297028.1¦ SARS coronavirus ZJ01,gi¦30421451¦gb¦AY282752.1¦ SARS coronavirus CUHK-Su10,gi¦34482146¦gb¦AY304495.1¦ SARS coronavirus GZ50,gi¦34482139¦gb¦AY304488.1¦ SARS coronavirus SZ16,gi¦34482137¦gb¦AY304486.1¦ SARS coronavirus SZ3,gi¦30027610¦gb¦AY278554.2¦ SARS coronavirus CUHK-W1,gi¦31416306¦gb¦AY279354.2¦ SARS coronavirus BJ04,gi¦37576845¦gb¦AY427439.1¦ SARS coronavirus AS,gi¦37361915¦gb¦AY283798.2¦ SARS coronavirus Sin2774,gi¦31416290¦gb¦AY278489.2¦ SARS coronavirus GD01,gi¦30468042¦gb¦AY283794.1¦ SARS coronavirus Sin2500,gi¦30468043¦gb¦AY283795.1¦ SARS coronavirus Sin2677,gi¦30468044¦gb¦AY283796.1¦ SARS coronavirus Sin2679,gi¦30468045¦gb¦AY283797.1¦ SARS coronavirus Sin2748,gi¦31982987¦gb¦AY286320.2¦ SARS coronavirus isolate ZJ-HZ01,gi¦30275666¦gb¦AY278488.2¦ SARS coronavirus BJ01.

SARS-CoV may be productive or replication defective. A “productive”SARS-CoV refers to a SARS-CoV particle that is capable of replication.The term “replication” includes, but is not limited to, the steps ofadsorbing (e.g., receptor binding) to a cell, entry into a cell (such asby endocytosis), introducing its genome sequence into the cell,uncoating the viral genome, initiating transcription of SARS-CoV genomicRNA to produce sgRNA, directing expression of SARS-CoV encapsidationproteins, encapsidating of the replicated viral nucleic acid sequencewith the encapsidation proteins into a viral particle that is releasedfrom the cell to infect other cells that are of either a permissive orsusceptible character. The terms “replication defective,”“replication-incompetent,” and “defective” SARS-Cov refer to a SARS-CoVparticle which is substantially incapable of one or more of the steps ofreplication.

The origin of SARS-CoV has not been determined but its emergence may bethe result of zoonotic transmission. The location and source of theSARS-CoV outbreak are reminiscent of influenza pandemics that havekilled millions of people in the past.

SARS-CoV has been isolated from humans, civet cats and a raccoon-dog,and has been propagated in kidney cells derived from different speciesof monkey (Drosten, et al., 2003, N. Engl. J. Med. 348:1967-1976;Ksiazek, et al., 2003, N. Engl. J. Med. 348:1953-1966; Peiris, et al.,2003, Lancet 361:1319-1325; Poutanen, et al., 2003, N. Engl. J. Med.348:1995-2005). Coronaviruses typically demonstrate a narrow host rangeand are species specific yet, SARS-CoV appears to have a broad hostrange. Furthermore, while disease caused by the known humanscoronaviruses is mild, SARS-CoV, like some of the animal coronaviruses,causes fatal disease (Holmes, et al., 1996, supra).

C. Cytophathic Effect Does Not Always Correlate With SARS-CoV Infection

Cell lines are routinely utilized to screen clinical specimen forrespiratory pathogens. At the beginning of the SARS-CoV outbreak,clinical specimen were inoculated onto panels of cells to identify thecausative agent of SARS (Drosten, et al., 2003, N. Engl. J. Med.348:1967-1976; Ksiazek, et al., 2003, N. Engl. J. Med. 348:1953-1966;Peiris, et al., 2003, Lancet 361:1319-1325). Based on CPE, Vero E6 andFRhMK cells were identified as susceptible to SARS-CoV infection(Drosten, et al., 2003, N. Engl. J. Med. 348:1967-1976; Ksiazek, et al.,2003, N. Engl. J. Med. 348:1953-1966; Peiris, et al., 2003, Lancet361:1319-1325). Surprisingly, however, coronaviruses can establishpersistent infection in cells without inducing CPE, suggesting that CPEmay not be an accurate indicator of infection (Chaloner, et al., 1981,Arch. Virol. 69:117-129). Data herein confirmed this surprisingobservation by demonstrating replication of SARS-CoV in the absence ofCPE. For example, significant CPE was not observed in pRhMK or pCMK 5days p.i., although the inventors discovered that virus titers wereactually increased within 24 hours p.i. (FIG. 2B, Table 1). TABLE 1Susceptibility of cells to SARS-CoV Species of SARS-CoV Viral Titer atCell Origin Replication CPE 48 hr (TCID₅₀) Vero E6 African green + + 2.4× 10⁷ monkey kidney pRhMK Primary rhesus + − 5.6 × 10⁵ monkey kidneypCMK Primary + − 7.8 × 10⁴ cynomologous monkey kidney MRC-5 Human lung −− 0 fibroblast MDCK Canine kidney − − N/D AK-D Feline lung − − N/Depithelia HRT-18 Human rectal − − 0 tumor L2 Murine fibroblast − − N/DR-Mix Mink and + − 7.8 × 10³ Human lung Mv1Lu Mink lung + − 2.5 × 10⁴A549 Human lung − − N/D epithelia HEL Human − − 0 embryonic lungHEK-293T Human + − 5.6 × 10³ embryonic kidney Huh-7 Human liver + − 1.3× 10⁵ CMT-93 Murine epithelia − − N/D CMT-93/hAPN Murine epithelia − −N/D BHK Baby hamster − − N/D kidney BHK/hAPN Baby hamster − − N/D kidneyD. SARS-CoV Does Not Bind to the Group 1 Coronavirus ReceptorAminopeptidase N (APN/CD13) and the Group 2 Coronavirus ReceptorCarcinoembryonic Antigen (CEACAM1a)

Aminopeptidase N, the receptor for group 1 coronaviruses is expressed onthe surface of epithelial cells of the kidney. The identification ofmonkey kidney cells susceptible to SARS-CoV, the culturing of SARS-CoVfrom the kidney of an infected patient, and sequence-based studiespredicting that the SARS-CoV Spike glycoprotein contains APN bindingdomains, led to the proposal that APN is a potential receptor forSARS-CoV (Yu, et al., 2003, Acta Pharmacol. Sin. 24:481-488). Based onthese assumptions, APN inhibitors were proposed for the treatment ofSARS-CoV infection (Kontoyiannis, et al., 2003, Lancet 361:1558).However, data herein (such as Examples 4 and 7) show that cellsexpressing species specific APN molecules as well as feline APN, theuniversal group 1 receptor, were all non permissive to SARS-CoV. Evencells expressing high levels of hAPN, previously demonstrated to besusceptible to HCoV-229E, were non permissive to SARS-CoV, suggestingthat SARS-CoV uses a receptor other than APN (Wentworth et al. 2001. J.Virol. 75:9741-9752.). Snijder et al. has suggested that SARS-CoV ismost closely related to Group II coronaviruses, suggesting that it mayuse a receptor utilized by a group 2 coronavirus. However, cell linespermissive to group 2 coronaviruses were not susceptible to SARS-CoV.Murine cells expressing CEACAM1a, the receptor for MHV and HRT-18 cellswhich are susceptible to HCoV-0C43, were both non permissive to SARS-CoVinfection. The inventors' findings suggest that SARS-CoV utilizes a yetunidentified receptor.

E. Cells Permissive to SARS-CoV

SARS-CoV was first isolated in African green monkey kidney cells (VeroE6) and fetal rhesus monkey kidney cells (FRhMK) inoculated withclinical specimen (Drosten, et al., 2003, N. Engl. J. Med.348:1967-1976; Ksiazek, et al., 2003, N. Engl. J. Med. 348:1953-1966;Peiris, et al., 2003, Lancet 361:1319-1325; Poutanen, et al., 2003, N.Engl. J. Med. 348:1995-2005). Based on cytopathic effect (CPE), othercells routinely used for identification of respiratory pathogens weredetermined to be non-permissive to SARS-CoV infection, such as MDCK,A549, NCI-H292, HeLa, LLC-MK2, Hut-292, B95-8, MRC-5, RDE and Hep-2(Drosten, et al., 2003, N. Engl. J. Med. 348:1967-1976; Ksiazek, et al.,2003, N. Engl. J. Med. 348:1953-1966; Peiris, et al., 2003, Lancet361:1319-1325).

To identify cell lines permissive to SARS-CoV, a multiplex reversetranscriptase polymerase chain reaction (RT-PCR) assay for detection ofSARS-CoV replication was developed by the inventors as described herein(Example 2). Primary cells and continuous cell lines derived from anumber of species and tissues were analyzed for susceptibility toSARS-CoV. Additionally, cells routinely used by clinical virologylaboratories for pathogen screening were analyzed for susceptibility toSARS-CoV. Data herein demonstrates the identification of identified bothhuman and non-human (monkey and mink) cells that support SARS-CoVreplication (Examples 3, 5, and 6, Table 1).

In particular, data herein (Example 3, Table 1) show that kidney cellsderived from three different species of monkey (African green monkey,Rhesus macaque and Cynomolgous macaque) were susceptible to productiveSARS-CoV infection. However, infection of pCMK and pRhMK cells resultedin lower viral titers than infection of Vero E6 cells. Without intendingto limit the invention to any particular mechanism, and while anunderstanding of the mechanism of the invention is not required, it isthe inventors' consideration that the discrepancy in virus productionmay be due to Vero E6 cells being a transformed cell line while pCMK andpRhMK are both primary cell populations. Furthermore, pCMK and pRhMK areboth mixed cell populations; the cells susceptible to SARS-CoV may makeup only a small percentage of the total cell population. Thus, incertain embodiments, it may be more advantageous to use cell lines suchas HEK-293T, Huh-7 and Mv1Lu cells as compared to primary cells such aspCMK and pRhMK.

Kuiken et al. recently demonstrated that Cynomolgous Macaques inoculatedwith SARS-CoV develop clinical symptoms similar to those observed ininfected humans. SARS-CoV was subsequently isolated from the inoculatedmonkeys (Fouchier, et al., 2003, Nature 423:240; Kuiken, et al., 2003,Lancet 362:263-270). However, SARS-CoV was not detected in kidney fromthese animals by immunohistochemical techniques. Surprisingly,therefore, and in contrast to Kuiken et al.'s report, the inventors'data suggest that kidney cells from monkeys supports SARS-CoVreplication (Ksiazek, et al., 2003, N. Engl. J. Med. 348:1953-1966).

Data herein (Example 5, Table 1) also identifies mink lung cells (Mv1Lu)as susceptible to SARS-CoV productive infection. In contrast, all of theclinically relevant cells that were tested by the inventors were notsusceptible to SARS-CoV infection. Mv1Lu cells are incorporated intorespiratory panels, that are used to screen clinical specimen forrespiratory pathogens including influenza A and B, adenovirus, RSV andparainfluenza. Additionally, Mv1Lu cells are a component of R-Mix, thecell mix which is also used for detection of RSV and parainfluenzaviruses. Without intending to limit the invention to any mechanism, andwhile an understanding of the mechanism of the invention is notnecessary, it is the inventors' view that the low level of virusreplication detected in Mv1Lu cells by titration may be due to slowerreplication of SARS-CoV in mink-derived cells than in Vero E6 cellswhere the virus was passaged.

Data herein (Example 6, Table 1) further demonstrates that twohuman-derived cell lines are susceptible to SARS-CoV productiveinfection. A kidney cell line (HEK-293T) and a liver cell line (Huh-7)were both permissive to SARS-CoV infection. HEK-293T cells weresusceptible to SARS-CoV infection but do not support production of hightiters of virus, suggesting that these cells may contain a block toSARS-CoV replication. Conversely, Huh-7 cells produced higher titers ofSARS-CoV although the titers were still lower then those produced frominfected Vero E6 cells. Without intending to limit the invention to anymechanism, and while an understanding of the mechanism of the inventionis not necessary, it is the inventors' opinion that the discrepancy inviral titers may be due to the passage of the virus in Vero E6 cellswhere it may have adapted.

In one embodiment, the cells used in any one of the invention's methodsis chosen from one or more of HEK-293T, Huh-7, Mv1Lu, pRHMK and pCMK.

While the invention is illustrated using HEK-293T, Huh-7, Mv1Lu, pRHMKand pCMK cells, it should be understood that the invention is notlimited to these particular cells, but rather includes equivalent cellsthat are established from these particular cells.

The term “established from” when made in reference to any cell disclosedherein (such as HEK-293T, Huh-7, Mv1Lu, pRHMK and/or pCMK cell, etc.)refers to a cell which has been obtained (e.g., isolated, purified,etc.) from the parent cell in issue using any manipulation, such as,without limitation, infection with virus, transfection with DNAsequences, treatment and/or mutagenesis using for example chemicals,radiation, etc., selection (such as by serial culture) of any cell thatis contained in cultured parent cells. For example, the inventionincludes within its scope cell lines that may be established from anycell disclosed herein (such as HEK-293T, Huh-7, Mv1Lu, pRHMK and/or pCMKcell, etc.) by treatment with chemical compounds (e.g.,N-ethyl-N-nitrosurea (ENU), methylnitrosourea (MNU), procarbazinehydrochloride (PRC), triethylene melamine (TEM), acrylamide monomer(AA), chlorambucil (CHL), melphalan (MLP), cyclophosphamide (CPP),diethyl sulfate (DES), ethyl methane sulfonate (EMS), methyl methanesulfonate (MMS), 6-mercaptopurine (6MP), mitomycin-C (MMC), procarbazine(PRC), N-methyl-N′-nitro-N-nitrosoguanidine (MNNG), ³H₂O, and urethane(UR)), and electromagnetic radiation (e.g., X-ray radiation,gamma-radiation, ultraviolet light).

In one embodiment, equivalent cells within the scope of the inventioninclude cells that are established from the exemplary HEK-293T, Huh-7,Mv1Lu, pRHMK and/or pCMK cells, and that have substantially the samesensitivity, increased sensitivity, or reduced sensitivity to SARS-CoVas the cell from which it is established. The term “sensitivity” and“sensitive” when made in reference to a cell is a relative term whichrefers to the degree of permissiveness of the cell to a virus ascompared to the degree of permissiveness of another cell to the samevirus. For example, the term “increased sensitivity” to SARS-CoV whenused in reference to the sensitivity of a first cell relative to asecond cell refers to an increase in the first cell, preferably at leasta 5%, more preferably from 5% to 10,000%, more preferably from 5% to1,000%, yet more preferably from 10% to 200%, and even more preferablyfrom 10% to 100%, increase in the quantity of SARS-CoV protein, SARS-CoVnucleic acid, and/or of CPE by progeny virus which is produced followinginfection of the first cell with SARS-CoV, as compared with the quantityof SARS-CoV protein, SARS-CoV nucleic acid, and/or of CPE by progenyvirus (respectively) which is produced following infection of the secondcell. For example, if 34 samples containing SARS-CoV were tested for thepresence of progeny virus, with 25 and 13 samples showing the presenceof CPE using a first cell and second cell, respectively, then thesensitivity is 74% and 38% for the first cell and second cell,respectively. This reflects an increase of 90% in the sensitivity of thefirst cell as compared to the sensitivity of the second cell.

In another embodiment, equivalent cells within the scope of theinvention include cells that are established from the exemplaryHEK-293T, Huh-7, Mv1Lu, pRHMK and/or pCMK cells, and that havesubstantially the same sensitivity to sARS-CoV as the cell from which itis established. This may be advantageous where, for example, the parentcell is made transgenic for a reporter gene.

In a further embodiment, equivalent cells within the scope of theinvention include cells that are established from the exemplaryHEK-293T, Huh-7, Mv1Lu, pRHMK and/or pCMK cells, and that have increasedsensitivity or decreased sensitivity to SARS-CoV as compared toHEK-293T, Huh-7, Mv1Lu, pRHMK and/or pCMK cells from which they wereestablished. This may be desirable where, for example, the parent cellis made transgenic for a receptor gene which alters the level of bindingof SARS-CoV to the cell.

The invention's cells (such as the exemplary HEK-293T, Huh-7, Mv1Lu,pRHMK and/or pCMK cell, etc.) show the surprising property of beingsusceptible to, and permissive for, infection by SARS-CoV. The term“susceptible” as used herein in reference to a cell describes theability of a permissive or non-permissive host cell to be infected by avirus. “Infection” refers to adsorption of the virus to the cell andpenetration into the cell. A cell may be susceptible without beingpermissive in that it can be penetrated by a virus in the absence ofviral replication and/or release of virions from the cell. A permissivecell line however must be susceptible. Susceptibility of a cell to avirus may be determined by methods known in the art such as detectingthe presence of viral proteins using electrophoretic analysis (i.e.,SDS-PAGE) of protein extracts prepared from the infected cell cultures.Susceptibility to SARS-CoV may also be determined by detecting thepresence of SARS-CoV gRNA using the exemplary methods disclosed herein.

The terms “permissive” and “permissiveness” as used herein describe thesequence of interactive events between a virus and its putative hostcell. The process begins with viral adsorption to the host cell surfaceand ends with release of infectious virions. A cell is “permissive”(i.e., shows “permissiveness”) if it is capable of supporting viralreplication as determined by, for example, production of viral nucleicacid sequences and/or of viral peptide sequences, regardless of whetherthe viral nucleic acid sequences and viral peptide sequences areassembled into a virion. While not required, in one embodiment, a cellis permissive if it generates virions and/or releases the virionscontained therein. Many methods are available for the determination ofthe permissiveness of a given cell line. For example, the replication ofa particular virus in a host cell line may be measured by the productionof various viral markers including viral proteins, viral nucleic acid(including both RNA and DNA) and the progeny virus. The presence ofviral proteins may be determined using electrophoretic analysis (i.e.,SDS-PAGE) of protein extracts prepared from the infected cell cultures.Viral nucleic acid may be quantitated using nucleic acid hybridizationassays. In a preferred embodiment, permissivity to SARS-CoV may also bedetermined by detecting the presence of SARS-CoV sgRNA using theexemplary methods disclosed herein. Susceptibility to SARS-CoV may alsobe determined by detecting the presence of SARS-CoV gRNA using theexemplary methods disclosed herein. Production of progeny virus may alsobe determined by observation of a cytopathic effect. However, thismethod is less preferred than detection of SARS-CoV sgRNA, since dataherein shows that a cytopathic effect may not be observed even whenviral replication is detectable by sgRNA (Table 1). The invention is notlimited to the specific quantity of replication of virus.

The terms “not permissive” and “non-infections” encompasses, forexample, a cell that is not capable of supporting viral replication asdetermined by, for example, production of viral nucleic acid sequencesand/or of viral peptide sequences, and/or assembly of viral nucleic acidsequences and viral peptide sequences into a virion.

The terms “cytopathic effect” and “CPE” as used herein describe changesin cellular structure (i.e., a pathologic effect). Common cytopathiceffects include cell destruction, syncytia (i.e., fused giant cells)formation, cell rounding, vacuole formation, and formation of inclusionbodies. CPE results from actions of a virus on permissive cells thatnegatively affect the ability of the permissive cellular host to preformits required functions to remain viable. In in vitro cell culturesystems, CPE is evident when cells, as part of a confluent monolayer,show regions of non-confluence after contact with a specimen thatcontains a virus. The observed microscopic effect is generally focal innature and the foci are initiated by a single virion. However, dependingupon viral load in the sample, CPE may be observed throughout themonolayer after a sufficient period of incubation. Cells demonstratingviral induced CPE usually change morphology to a rounded shape, and overa prolonged period of time can die and be released from their anchoragepoints in the monolayer. When many cells reach the point of focaldestruction, the area is called a viral plaque, which appears as a holein the monolayer. The terms “plaque” and “focus of viral infection”refer to a defined area of CPE which is usually the result of infectionof the cell monolayer with a single infectious virus which thenreplicates and spreads to adjacent cells of the monolayer. Cytopathiceffects are readily discernable and distinguishable by those skilled inthe art.

In another embodiment, the invention contemplates the use of transgeniccells such as transgenic HEK-293T, transgenic Huh-7, transgenic Mv1Lu,transgenic pRHMK and/or transgenic pCMK cells. The terms “transgenic”and “genetically engineered” when made in reference to a cell, refer toa cell that has been transformed to contain a transgene. The term“transformation” as used herein refers to the introduction of atransgene into a cell by way of human intervention, using standardmethods in the art. For example, where the nucleic acid sequence is aplasmid or naked piece of linear DNA, the sequence may be “transfected”into the cell using, for example, calcium phosphate-DNAco-precipitation, DEAE-dextran-mediated transfection, polybrene-mediatedtransfection, electroporation, microinjection, liposome fusion,lipofection, protoplast fusion, and biolistics. Alternatively, where thenucleic acid sequence is encapsidated into a viral particle, thesequence may be introduced into a cell by “infecting” the cell with thevirus.

Transformation of a cell may be stable or transient. The terms“transient transformation” and “transiently transformed” refer to theintroduction of one or more nucleotide sequences of interest into a cellin the absence of integration of the nucleotide sequence of interestinto the host cell's genome. Transient transformation may be detectedby, for example, enzyme-linked immunosorbent assay (ELISA) which detectsthe presence of a polypeptide encoded by one or more of the nucleotidesequences of interest. Alternatively, transient transformation may bedetected by detecting the activity of the protein (e.g.,β-glucuronidase) encoded by the nucleotide sequence of interest. Theterm “transient transformant” refer to a cell which has transientlyincorporated one or more nucleotide sequences of interest. Transienttransformation with the invention's vectors may be desirable in, forexample, cell biology or cell cycle investigations which requireefficient gene transfer.

In contrast, the terms “stable transformation” and “stably transformed”refer to the introduction and integration of one or more nucleotidesequence of interest into the genome of a cell. Thus, a “stabletransformant” is distinguished from a transient transformant in that,whereas genomic DNA from the stable transformant contains one or morenucleotide sequences of interest, genomic DNA from the transienttransformant does not contain the nucleotide sequence of interest.Stable transformation of a cell may be detected by Southern blothybridization of genomic DNA of the cell with nucleic acid sequenceswhich are capable of binding to one or more of the nucleotide sequencesof interest. Alternatively, stable transformation of a cell may also bedetected by the polymerase chain reaction of genomic DNA of the cell toamplify the nucleotide sequence of interest.

A transgene that is introduced into the cells of the invention maycomprise nucleotide sequence that is “endogenous” or “heterologous”(i.e., “foreign”). The term “endogenous” refers to a sequence which isnaturally found in the cell or virus into which it is introduced so longas it does not contain some modification relative to thenaturally-occurring sequence. The term “heterologous” refers to asequence which is not endogenous to the cell or virus into which it isintroduced. For example, heterologous DNA includes a nucleotide sequencewhich is ligated to, or is manipulated to become ligated to, a nucleicacid sequence to which it is not ligated in nature, or to which it isligated at a different location in nature. Heterologous DNA alsoincludes a nucleotide sequence which is naturally found in the cell orvirus into which it is introduced and which contains some modificationrelative to the naturally-occurring sequence. Generally, although notnecessarily, heterologous DNA encodes heterologous RNA and heterologousproteins that are not normally produced by the cell or virus into whichit is introduced. Examples of heterologous DNA include reporter genes,transcriptional and translational regulatory sequences, DNA sequenceswhich encode selectable marker proteins (e.g., proteins which conferdrug resistance), etc.

The term “wild-type” when made in reference to a peptide sequence andnucleotide sequence refers to a peptide sequence and nucleotidesequence, respectively, which has the characteristics of that peptidesequence and nucleotide sequence when isolated from a naturallyoccurring source. A wild-type peptide sequence and nucleotide sequenceis that which is most frequently observed in a population and is thusarbitrarily designated the “normal” or “wild-type” form of the peptidesequence and nucleotide sequence, respectively. In contrast, the term“modified” or “mutant” refers to a peptide sequence and nucleotidesequence which displays modifications in sequence and/or functionalproperties (i.e., altered characteristics) when compared to thewild-type peptide sequence and nucleotide sequence, respectively. It isnoted that naturally-occurring mutants can be isolated; these areidentified by the fact that they have altered characteristics whencompared to the wild-type peptide sequence and nucleotide sequence.Nucleic acid sequences and/or proteins may be modified by chemical,biochemical, and/or molecular biological techniques. Modifications tonucleic acid sequences include introduction of one or more deletion,insertion, and substitution. A “deletion” is defined as a change in anucleic acid sequence in which one or more nucleotides is absent. An“insertion” or “addition” is that change in a nucleic acid sequencewhich has resulted in the addition of one or more nucleotides. A“substitution” results from the replacement of one or more nucleotidesby a molecule which is a different molecule from the replaced one ormore nucleotides.

While not required, in one embodiment, it may be desirable that thetransgene contains a sequence encoding a selectable marker. The term“selectable marker” as used herein refers to nucleotide sequence whichencodes an enzymatic activity that confers resistance to a compound(e.g., antibiotic or drug) upon the cell in which the selectable markeris expressed. Selectable markers may be “positive”; i.e., genes whichencode an enzymatic activity which can be detected in any cell or cellline. Examples of dominant selectable markers include, but are notlimited to, (1) the bacterial aminoglycoside 3′ phosphotransferase gene(also referred to as the neo gene) which confers resistance to the drugG418 in cells, (2) the bacterial hygromycin G phosphotransferase (hyg)gene which confers resistance to the antibiotic hygromycin, and (3) thebacterial xanthine-guanine phosphoribosyl transferase gene (alsoreferred to as the gpt gene) which confers the ability to grow in thepresence of mycophenolic acid. Other selectable markers are not dominantin that their use must be in conjunction with a cell line that lacks therelevant enzyme activity. Selectable markers may be “negative”; negativeselectable markers encode an enzymatic activity whose expression iscytotoxic to the cell when grown in an appropriate selective medium. Forexample, the HSV-tk gene and the dt gene are commonly used as a negativeselectable marker. Expression of the HSV-tk gene in cells grown in thepresence of gancyclovir or acyclovir is cytotoxic; thus, growth of cellsin selective medium containing gancyclovir or acyclovir selects againstcells capable of expressing a functional HSV TK enzyme. Similarly, theexpression of the dt gene selects against cells capable of expressingthe Diphtheria toxin. In one embodiment, the selectable marker gene usedis the neo gene in plasmid pcDNA3 (Invitrogen) and cells whichincorporat this transgene may be selected by exposure to Geneticin(G418) (Gibco-BRL Inc.).

In another embodiment, it may be desirable that the transgene contains asequence (e.g., the uid A gene) encoding a reporter protein. This may bedesirable where, for example, the reporter protein is more readilydetectable than another protein to which it is operably linked. The term“reporter gene” refers to a gene which encodes a reporter molecule(e.g., RNA, polypeptide, etc.) which is detectable in any detectionsystem, including, but not limited to enzyme (e.g., ELISA, as well asenzyme-based histochemical assays), fluorescent, radioactive, andluminescent systems. Exemplary reporter genes include, for example,β-glucuronidase gene, green fluorescent protein gene, E. coliβ-galactosidase (LacZ) gene, Halobacterium β-galactosidase gene, E. coliluciferase gene, Neuropsora tyrosinase gene, Aequorin (jellyfishbioluminescenece) gene, human placental alkaline phosphatase gene, andchloramphenicol acetyltransferase (CAT) gene. Reporter genes arecommercially available, such as from Clontech, Invtrogen, and Promega.It is not intended that the present invention be limited to anyparticular detection system or label.

In a further embodiment, it may be desirable that the transgenic cell(such as transgenic cells of the exemplary HEK-293T, Huh-7, Mv1Lu, pRHMKand/or pCMK cell, etc.) expresses a probe gene. The term “probe” generefers to a sequence useful in the detection, identification and/orisolation of particular polypeptide sequence. Exemplary probe genesencode ligand-binding systems useful for the isolation of polypeptidessuch as the staphylococcal protein A and its derivative ZZ (which bindsto human polyclonal IgG), histidine tails (which bind to Ni²⁺), biotin(which binds to streptavidin), maltose-binding protein (MBP) (whichbinds to amylose), glutathione S-transferase (which binds toglutathione), etc. Exemplary probe gene sequences include reportergenes, as discussed above.

In yet another embodiment, the transgenic cell (such as the transgeniccell of an exemplary HEK-293T, Huh-7, Mv1Lu, pRHMK and/or pCMK cell,etc.) may express a “fusion protein.” Exemplary sequences that may beincluded in a fusion gene include those for adenosine deaminase (ADA)gene (GenBank Accession No. M13792); alpha-1-antitrypsin gene (GenBankAccession No. M11465); beta chain of hemoglobin gene (GenBank AccessionNo. NM_(—)000518); receptor for low density lipoprotein gene (GenBankAccession No. D16494); lysosomal glucocerebrosidase gene (GenBankAccession No. K02920); hypoxanthine-guanine phosphoribosyltransferase(HPRT) gene (GenBank Accession No. M26434, J00205, M27558, M27559,M27560, M27561, M29753, M29754, M29755, M29756, M29757); lysosomalarylsulfatase A (ARSA) gene (GenBank Accession No. NM_(—)000487);ornithine transcarbamylase (OTC) gene (GenBank Accession No.NM_(—)000531); phenylalanine hydroxylase (PAH) gene (GenBank AccessionNo. NM_(—)000277); purine nucleoside phosphorylase (NP) gene (GenBankAccession No. NM_(—)000270); the dystrophin gene (GenBank Accession Nos.M18533, M17154, and M18026); the utrophin (also called the dystrophinrelated protein) gene (GenBank Accession No. NM_(—)007124); and thehuman cystic fibrosis transmembrane conductance regulator (CFTR) gene(GenBank Accession No. M28668).

In another embodiment, the transgenic cell (such as the transgenic cellof the exemplary HEK-293T, Huh-7, Mv1Lu, pRHMK and/or pCMK cell, etc.)may be transfected with nucleotide sequences encoding a cytokine.“Cytokine” refers to a molecule, such a protein or glycoprotein,involved in the regulation of cellular proliferation and function.Cytokines are exemplified by lymphokines (e.g., tumor necrosis factor-α(TNF-α), tumor necrosis factor-β (TNF-β), tumor necrosis factor-γ(TNF-γ), etc.), interferons such as interferon-γ (IFN-γ), tumor necrosisfactor (TNF), etc.), growth factors (e.g., erythropoietin, G-CSF, M-CSF,GM-CSF, epidermal growth factor (EGF), insulin, platelet-derived growthfactor (PDGF), fibroblast growth factor (FGF), etc.), and interleukins(e.g., interleukin-2 (IL-2), interleukin-4 (IL-4), interleukin-5 (IL-5),interleukin-6 (IL-6), interleukin-9 (IL-9), interleukin-10 (IL-10), andinterleukin-13 (IL-13)).

The transgenic cells may be useful where it is desirable to determinethe effect of the transgenic polypeptide on the cell's susceptibilityand/or permissivity to SARS-CoV. For example, increased permissivity ofthe transgenic cell compared to the cell into which the transgene wasintroduced may be useful in generating higher virus titers and/or higherviral proteins for use in vaccine production and/or generation ofantibodies. Conversely, reduced permissivity of the transgenic cellcompared to the cell into which the transgene was introduced may beuseful in reducing the risk of infection with SARS-CoV. For example,Mv1Lu cells are routinely used in diagnostic assays for the detection ofinfluenza and/or parainfluenza viruses. Thus, a transgenic Mv1Lu cellwith reduced permissivity to SARS-CoV compared to a Mv1Lu cell intowhich the transgene was introduced is safer to use in small laboratoriesfor detection of influenza and/or parainfluenza viruses without the needto resort to containment approaches that would otherwise be required forcells producing infectious SARS-CoV. Thus, in one embodiment, the Mv1Lucells (whether or not they are transgenic) retain their susceptibilityto one or more of influenza virus and parainfluenza virus.

In a further embodiment, the transgenic cell (such as the transgeniccell of the exemplary HEK-293T, Huh-7, Mv1Lu, pRHMK and/or pCMK cell,etc.) may be engineered to include other nucleotide sequences ofinterest, such as non-coding regulatory sequences which do not encode anmRNA or protein product, (e.g., promoter sequence, polyadenylationsequence, termination sequence, enhancer sequence, etc.). Exemplary“promoters” include, without limitation, single, double and triplepromoters.

In a further embodiment, the transgenic cell (such as the transgeniccell of the exemplary HEK-293T, Huh-7, Mv1Lu, pRHMK and/or pCMK cell,etc.) expresses a receptor gene. The term “receptor” refers to astructure (generally, but not necessarily, a protein) located on or in acell, which specifically recognizes a binding molecule (i.e., a ligand).In one embodiment, this binding initiates either a specific biologicalresponse or the transduction of a signal. However, it is not necessarythat binding result is in a specific biological response or thetransduction of a signal, as for example when a virus binds to areceptor on a cell.

In one embodiment, the transgenic cell is a Mv1Lu-hF cell (ATCCaccession No. PTA-4737), i.e., a transgenic Mv1Lu that expresses humanfurin, as described in U.S. Pat. No. 6,610,474, which is incorporated byreference in its entirety.

In a further embodiment, the transgenic cell comprises a cell lineestablished from a transgenic cell line designated Mv1Lu-hF, wherein theestablished cell line has a property selected from the group consistingof (a) increased sensitivity to at least one virus selected from thegroup consisting of influenza A virus, influenza B virus andparainfluenza virus 3, as compared to the Mv1Lu cell line, and (b)enhanced productivity of infectious virions upon inoculation with atleast one virus selected from the group one consisting of influenza Avirus, influenza B virus and parainfluenza virus 3, as compared to theMv1Lu cell line. In a more preferred embodiment, the cell line has thesensitivity of the cell line designated Mv1Lu-hF, to at least one virusselected from the group consisting of influenza A virus, influenza Bvirus and parainfluenza virus 3. In an alternative embodiment, thetransgenic cell comprises a transgenic mink lung epithelial cell lineexpressing human furin, wherein the cell line has a property selectedfrom the group consisting of (a) increased sensitivity to at least onevirus selected from the group consisting of influenza A virus, influenzaB virus and parainfluenza virus 3, as compared to Mv1Lu, and (b)enhanced productivity of infectious virions upon inoculation with atleast one virus selected from the group one consisting of influenza Avirus, influenza B virus and parainfluenza virus 3, as compared toMv1Lu. More preferably, transgenic mink lung epithelial cell line hasthe sensitivity of the cell line designated Mv1Lu-hF and deposited asATCC accession number PTA-4737, to at least one virus selected from thegroup consisting of influenza A virus, influenza B virus andparainfluenza virus 3. Each of these transgenic cells is described inU.S. Pat. No. 6,610,474, which is incorporated by reference in itsentirety

F. Exemplary Assays for Detecting Replication of SARS-CoV

The invention provides a method for detecting replication ofSARS-coronavirus in a sample, comprising detecting the presenceSARS-coronavirus sgRNA in the sample. These methods are useful in, forexample, diagnosing the presence of SARS-CoV, identifying cells that aresusceptible and/or permissive for SARS-CoV, screening agents that alterinfection with SARS-CoV, and in determining the relative efficacy ofagents and/or modalities of treatment in altering SARS infection.

One aspect that distinguishes the invention's methods from the prior artis that the invention's methods detect the presence of sgRNA, whereasprior art methods relied on detection of gRNA, which only detected virusinput, but could not distinguish this from viral RNA replication andmRNA production (Drosten, et al., 2003, N. Engl. J. Med. 348:1967-1976;Poon, et al., 2003, Clin. Chem. 49:953-955; Poutanen, et al., 2003, N.Engl. J. Med. 348:1995-2005). Thus, the invention's assay candifferentiate non-replicating genomic SARS-CoV RNA from the replicativeforms produced during an active infection. The assay could therefore beused to differentiate exposure (or mechanical transmission in an animalvector) from active infection and or viral replication.

Also, the invention's methods that utilize detection of sgRNA aredistinguished from prior methods that use CPE for detection of virus inthat data herein (Table 1) confirms that CPE is not an accurateindicator of SARS-CoV replication, whereas detection of sgRNAreproducibly detected such replication. The invention's use of sgRNA toidentify an active SARS-CoV infection is not currently used to diagnosehuman and/or animal coronaviruses of veterinary importance.

The invention's methods are also distinguished from methods using RACEassay (Zeng et al. (2003) Exp. Biol. Med. 228(7):866-73).

The detection of sgRNA in accordance with the invention's methods detectearly replication of SARS-CoV. One utility and advantage of this methodis that, when coupled with titration of viral supernatants, cellspermissive to SARS-CoV can be identified. As disclosed herein (Examples3, 5 and 6), the invention's methods have successfully identifiedmonkey, mink and human cells that are susceptible and permissive toSARS-CoV infection. The finding that SARS-CoV enters various cell typesand initiates replication is useful as the basis for the development ofdiagnostic assays, especially when coupled with a SARS-CoV-specificnucleic acid and/or SARS-CoV-specific antigen detection methods. WhereRNA replication results in the production of infectious virions, thepermissive cell lines are also useful candidates for vaccine production.Identification of cell lines that result in abortive replication willlead to more sensitive and/or safer diagnostic cells that can be used asantigen sources and for identifying potential anti-SARS-CoV drugtargets.

Additionally, cells that are susceptible to SARS-CoV binding and entry,but that have blocks between the initiation of replication and theproduction of new virus, can also be identified using the invention'smethods. The use of molecular diagnostic methods such as nucleic acidprobes and monoclonal antibodies has however, demonstrated thatnon-permissive cells may have the ability to provide a cell-based testfor detecting the presence of a virus. In fact, when consideringdetection of viruses that require level III biological containment forcell culture amplification, e.g. SARS coronavirus, a cell line that doesnot produce and release infectious virus to a high level may havesubstantial advantages in safety. An example from the art of thediagnostic use of a non-permissive cell line is the use of mink lungcells (Mv1Lu) to detect cytomegalovirus (CMV) (Gleaves, et al., J ClinMicrobiol, 1992. 30(4): p. 1045-8). Human embryonic lung cells (MRC-5)are considered the cell line of choice to produce infectious CMV virus,however mink lung cells have been shown to be useful in detecting theprimary infection event of CMV by using a monoclonal antibody thattargets the CMV immediate early (mIE Ag) protein that is produced inabundant amounts. Reported benefits of the non-permissive Mv1Lu cellsover MRC-5 cells were higher detection sensitivity and lower toxicityfrom non-specific material present in the clinical specimen. Mink lungcells have also been shown to be commercially useful for influenza virusdetection.

i. sgRNA

In one embodiment, the invention's methods detect sgRNA. The term“subgenomic RNA” and “sgRNA” are used interchangeably herein to refer toa nucleotide sequence comprising at least a portion of the leadersequence.

The term “leader sequence” refers to a sequence of about 40 to about150, about 50 to about 80, and or about 55 to about 75, nucleotides thatis located at the 5′ terminus of the genome. This sequence is juxtaposedto the 5′ terminus of each subgenomic RNA by transcriptional mechanismsduring synthesis. There is very strong sequence conservation of theleader sequence across the strains of SARS (Drosten et al., N. Engl. J.Med. 2003;1967-76; Ksiazek et al., N. Engl. J. Med. 2003;1953-66; Marraet al., Science 2003;1399-404). The leader sequence plays a role in thegeneration of the subgenomic RNA transcripts (Holmes et al., In: Knipe DM, Howley P M, Griffin D E, Lamb R A, Martin M A, Roizman B, eds. FieldsVirology. Philadelphia: Lippincott Williams & Wilkins, 2001;1187-1203;Holmes et al. “Coronaviridae: The viruses and their replication.” In:Fields B N, Knipe D M, Howely P M, eds. Fields Virology. Philadelphia:Lippincott-Raven, 1996;1075-1094; Sawicki et al., J. Gen. Virol.2001;385-96; Sawicki et al., Adv. Exp. Med. Biol. 1998;215-9; Wang etal., Adv. Exp. Med. Biol. 2001;491-7), transcription, replication,translation and/or packaging of viral RNA.

Sequence alignment by the inventors showed conservation of at least aportion of the leader sequence in the following exemplary strains ofSARS-CoV: gi¦31416292¦gb¦AY278487.3¦ SARS coronavirusBJ02,gi¦30248028¦gb¦AY274119.3¦ SARS coronavirus TOR2,gi¦30698326¦gb¦AY291451.1¦ SARS coronavirus TW1,gi¦33115118¦gb¦AY323977.2¦ SARS coronavirus HSR 1,gi¦35396382¦gb¦AY394850.1¦ SARS coronavirus WHU,gi¦33411459¦dbj¦AP006561.1¦ SARS coronavirus TWY,gi¦33411444¦dbj¦AP006560.1¦ SARS coronavirus TWS,gi¦33411429¦dbj¦AP006559.1¦SARS coronavirus TWK,gi¦33411414¦dbj¦AP006558.1¦ SARS coronavirus TWJ,gi¦33411399¦dbj¦AP006557.1¦ SARS coronavirus TWH,gi¦30023963¦gb¦AY278491.2¦ SARS coronavirus HKU-39849,gi¦33578015¦gb¦AY310120.1¦ SARS coronavirus FRA,gi¦33518725¦gb¦AY362699.1¦ SARS coronavirus TWC3,gi¦33518724¦gb¦AY362698.1¦ SARS coronavirus TWC2,gi¦30027617¦gb¦AY278741.1¦ SARS coronavirus Urbani,gi¦31873092¦gb¦AY321118.1¦ SARS coronavirus TWC,gi¦33304219¦gb¦AY351680.1¦ SARS coronavirus ZMY 1,gi¦31416305¦gb¦AY278490.3¦SARS coronavirus BJ03,gi¦30910859¦gb¦AY297028.1¦ SARS coronavirus ZJ01,gi¦30421451¦gb¦AY282752.1¦ SARS coronavirus CUHK-Su10,gi¦34482146¦gb¦AY304495.1¦ SARS coronavirus GZ50,gi¦34482139¦gb¦AY304488.1¦ SARS coronavirus SZ16,gi¦34482137¦gb¦AY304486.1¦ SARS coronavirus SZ3,gi¦30027610¦gb¦AY278554.2¦ SARS coronavirus CUHK-W1,gi¦31416306¦gb¦AY279354.2¦ SARS coronavirus BJ04,gi¦37576845¦gb¦AY427439.1¦ SARS coronavirus AS,gi¦37361915¦gb¦AY283798.2¦ SARS coronavirus Sin2774,gi¦31416290¦gb¦AY278489.2¦ SARS coronavirus GD01,gi¦30468042¦gb¦AY283794.1¦ SARS coronavirus Sin2500,gi¦30468043¦gb¦AY283795.1¦ SARS coronavirus Sin2677,gi¦30468044¦gb¦AY283796.1¦ SARS coronavirus Sin2679,gi¦30468045¦gb¦AY283797.1¦ SARS coronavirus Sin2748,gi¦31982987¦gb¦AY286320.2¦ SARS coronavirus isolate ZJ-HZ01,gi¦30275666¦gb¦AY278488.2¦ SARS coronavirus BJ01.

In one embodiment, the leader sequence is exemplified by the sequencefrom nucleotide 1 to nucleotide 72 for SARS-CoV (Urbani) (FIG. 7):5′-atattaggtttttacctacccaggaaaagccaaccaacctcgatctcttgtagatctgttctctaaacgaac-3′ (SEQ IDNO:36);5′-tattaggtttttacctacccaggaaaagccaaccaacctcgatctcttgtagatctgttctctaaacgaac-3′(SEQ ID NO:37) of gi¦33304219¦gb¦AY351680.1¦ SARS coronavirus ZMY1,5′-taggtttttacctacccaggaaaagccaaccaacctcgatctcttgtagatctgttctctaaacgaac-3′(SEQ ID NO:38) of gi¦31416305¦gb¦AY278490.3¦ SARS coronavirusBJ03,5′-ctacccaggaaaagccaaccaacctcgatctcttgtagatctgttctctaaacgaac-3′(SEQ ID NO:77) of gi¦30421451¦gb¦AY282752.1¦ SARS coronavirus CUHK-Su10,5′-tacccaggaaaagccaaccaacctcgatctcttgtagatctgttctctaaacgaac-3′ (SEQ IDNO:78) of gi¦31416306¦gb¦AY279354.2¦ SARS coronavirus BJ04, and5′-ccaggaaaagccaaccaacctcgatctcttgtagatctgttctctaaacgaac-3′ (SEQ IDNO:79) of gi¦30275666¦gb¦AY278488.2¦ SARS coronavirus BJ01.

As disclosed herein, portions of the leader sequence are expresslycontemplated as equivalents to the full length leader sequence for thedetection of sgRNA and/or SARS-CoV replication. Exemplary portions ofthe SARS-CoV (Urbani) leader sequence include, without limitation, 5′-5′-atattaggtttttacctacccaggaaaagccaaccaacctcgatctcttgtagatctgttct-3′,(SEQ ID NO:39)5′-atattaggtttttacctacccaggaaaagccaaccaacctcgatctcttgtagatct-3′, (SEQ IDNO:40) 5′-atattaggtttttacctacccaggaaaagccaaccaacctcgatctcttgtag3′, (SEQID NO:41) 5′-atattaggtttttacctacccaggaaaagccaaccaacctcgatc-3′, (SEQ IDNO:42) 5′-atattaggtttttacctacccaggaaaagccaaccaacc-3′, (SEQ ID NO:43)5′-atattaggtttttacctacccaggaaaagccaac-3′, (SEQ ID NO:44)5′-atattaggtttttacctacccaggaaaagc-3′, (SEQ ID NO:45)5′-atattaggtttttacctacccagg-3′, (SEQ ID NO:46)5′-atattaggtttttacctac-3′, (SEQ ID NO:47) 5′-atattagg-3′, (SEQ ID NO:48)5′-ttacctacccaggaaaagccaaccaacctcgatctcttgtagatctgttctctaaacgaac-3′,(SEQ ID NO:49) 5′-aaaagccaaccaacctcgatctcttgtagatctgttctctaaacgaac-3′,(SEQ ID NO:50) 5′-gccaaccaacctcgatctcttgtagatctgttctctaaacgaac-3′, (SEQID NO:51) 5′-ccaacctcgatctcttgtagatctgttctctaaacgaac-3′, (SEQ ID NO:52)5′-ctcgatctcttgtagatctgttctctaaacgaac-3′, (SEQ ID NO:53)5′-tcttgtagatctgttctctaaacgaac-3′, (SEQ ID NO:54)5′-gatctgttctctaaacgaac-3′, (SEQ ID NO:55) and 5′-taaacgaac-3′, (SEQ IDNO:56) 5′- atattaggtt tttacctacc caggaaaagc caaccaacct cgatctcttgtagatctgtt -3′, (SEQ ID NO:57) 5′- atattaggtt tttacctacc caggaaaagccaaccaacct cgatctcttg -3′, (SEQ ID NO:58) 5′- atattaggtt tttacctacccaggaaaagc caaccaacct -3′, (SEQ ID NO:59) 5′- atattaggtt tttacctacccaggaaaagc -3′, (SEQ ID NO:60) 5′- atattaggtt tttacctacc -3′, (SEQ IDNO:61) 5′- atattaggtt -3′, (SEQ ID NO:62) 5′- tttacctacc caggaaaagccaaccaacct cgatctcttg tagatctgtt ctctaaacga ac-3′, (SEQ ID NO:63) 5′-caggaaaagc caaccaacct cgatctcttg tagatctgtt ctctaaacga ac-3′, (SEQ IDNO:64) 5′- caaccaacct cgatctcttg tagatctgtt ctctaaacga ac-3′, (SEQ IDNO:65) 5′- cgatctcttg tagatctgtt ctctaaacga ac-3′, (SEQ ID NO:66) 5′-tagatctgtt ctctaaacga ac-3′, (SEQ ID NO:67) and 5′- ctctaaacga ac-3′.(SEQ ID NO:68)

In one embodiment, the sgRNA comprises at least a portion of a leadersequence operably linked to at least a portion of a gene encoding aSARS-CoV polypeptide. The term “polypeptide,” “protein,” “peptide,”“peptide sequence,” “amino acid sequence,” and “polypeptide sequence”are used interchangeably herein to refer to at least two amino acids oramino acid analogs which are covalently linked by a peptide bond or ananalog of a peptide bond. The term peptide includes oligomers andpolymers of amino acids or amino acid analogs. The term peptide alsoincludes molecules which are commonly referred to as peptides, whichgenerally contain from about two (2) to about twenty (20) amino acids.The term peptide also includes molecules which are commonly referred toas polypeptides, which generally contain from about twenty (20) to aboutfifty amino acids (50). The term peptide also includes molecules whichare commonly referred to as proteins, which generally contain from aboutfifty (50) to about three thousand (3000) amino acids. The amino acidsof the peptide may be L-amino acids or D-amino acids. A peptide,polypeptide or protein may be synthetic, recombinant or naturallyoccurring. A synthetic peptide is a peptide which is produced byartificial means in vitro (e.g., was not produced in vivo).

The term “SARS-CoV polypeptide” refers to any polypeptide that isencoded by the SARS-CoV genome (regardless of whether the genome is“wild type” or “modified”), including, for example, antigenicpolypeptides. SARS-CoV polypeptides are exemplified by, but not limitedto, Nucleocapsid (N), Spike glycoprotein (S), Matrix (M), E protein, andReplicase proteins (Pol 1a/b).

The “Nucleocapsid” protein (also referred to as “N”) refers to a proteinthat is produced early in infection and at very high abundance. The N ofother CoVs is highly immunogenic, eliciting antibodies and T-cellresponses in natural infections. The Nucleocapsid protein isexemplified, but not limited to, the sequences in FIGS. 23-26 and 40,and those encoded by the genomic sequences in gi¦31416292¦gb¦AY278487.3¦SARS coronavirus BJ02,gi¦30248028¦gb¦AY274119.3¦ SARS coronavirus TOR2,gi¦30698326¦gb¦AY291451.1¦ SARS coronavirus TW1,gi¦33115118¦gb¦AY323977.2¦ SARS coronavirus HSR 1,gi¦35396382¦gb¦AY394850.1¦ SARS coronavirus WHU,gi¦33411459¦dbj¦AP006561.1¦ SARS coronavirus TWY,gi¦33411444¦dbj¦AP006560.1¦ SARS coronavirus TWS,gi¦33411429¦dbj¦AP006559.1¦ SARS coronavirus TWK,gi¦33411414¦dbj¦AP006558.1¦ SARS coronavirus TWJ,gi¦33411399¦dbj¦AP006557.1¦ SARS coronavirus TWH,gi¦30023963¦gb¦AY278491.2° SARS coronavirus HKU-39849,gi¦33578015¦gb¦AY310120.1¦ SARS coronavirus FRA,gi¦33518725¦gb¦AY362699.1¦ SARS coronavirus TWC3,gi¦33518724¦gb¦AY362698.1¦ SARS coronavirus TWC2,gi¦30027617¦gb¦278741.1¦ SARS coronavirus Urbani,gi¦31873092¦gb¦AY321118.1¦ SARS coronavirus TWC,gi¦33304219¦gb¦AY351680.1¦ SARS coronavirus ZMY 1,gi¦31416305¦gb¦AY278490.3¦ SARS coronavirus BJ03,gi¦30910859¦gb¦AY297028.1¦ SARS coronavirus ZJ01,gi¦30421451¦gb¦AY282752.1¦ SARS coronavirus CUHK-Su10,gi¦34482146¦gb¦AY304495.1¦ SARS coronavirus GZ50,gi¦34482139¦gb¦AY304488.1¦ SARS coronavirus SZ16,gi¦34482137¦gb¦AY304486.1¦ SARS coronavirus SZ3,gi¦30027610¦gb¦AY278554.2¦ SARS coronavirus CUHK-W1,gi¦31416306¦gb¦AY279354.2¦SARS coronavirus BJ04,gi¦37576845¦gb¦AY427439.1¦ SARS coronavirus AS,gi¦37361915¦gb¦AY283798.2¦SARS coronavirus SIN2774,gi¦31416290¦gb¦AY278489.2¦ SARS coronavirus GD01,gi¦30468042¦gb¦AY283794.1¦ SARS coronavirus Sin2500,gi¦30468043¦gb¦AY283795.1¦ SARS coronavirus Sin2677,gi¦30468044¦gb¦AY283796.1¦ SARS coronavirus Sin2679,gi¦30468045¦gb¦AY283797.1¦ SARS coronavirus Sin2748,gi¦31982987¦gb¦AY286320.2¦ SARS coronavirus isolate ZJ-HZ01, andgi¦30275666¦gb¦AY278488.2¦ SARS coronavirus BJ01.

The “Spike glycoprotein” (also referred to as “S”) refers to a viralattachment protein that protrudes from the viron and that is a majorantigenic determinant. Antibodies to this protein may neutralize thevirus rendering it non infectious. The Spike glyocoprotein isexemplified, but not limited to, the sequences in FIGS. 27-29 and 41,and by those encoded by the genomic sequences ingi¦31416292¦gb¦AY278487.3¦ SARS coronavirusBJ02,gi¦30248028¦gb¦AY274119.3¦ SARS coronavirus TOR2,gi¦30698326¦gb¦AY291451.1¦ SARS coronavirus TW1,gi¦33115118¦gb¦AY323977.2¦ SARS coronavirus HSR 1,gi¦35396382¦gb¦AY394850.1¦ SARS coronavirus WHU,gi¦33411459¦dbj¦AP006561.1¦ SARS coronavirus TWY,gi¦33411444¦dbj¦AP006560.1¦ SARS coronavirus TWS,gi¦33411429¦dbj¦AP006559.1¦ SARS coronavirus TWK,gi¦33411414¦dbj¦AP006558.1¦ SARS coronavirus TWJ,gi¦33411399¦dbj¦AP006557.1¦ SARS coronavirus TWH,gi¦30023963¦gb¦AY278491.2¦ SARS coronavirus HKU-39849,gi¦33578015¦gb¦AY310120.1¦ SARS coronavirus FRA,gi¦33518725¦gb¦AY362699.1 ¦ SARS coronavirus TWC3,gi¦33518724¦gb¦AY362698¦ SARS coronavirus TWC2,gi¦30027617¦gb¦AY278741.1¦ SARS coronavirus Urbani,gi¦31873092¦gb¦AY321118.1¦ SARS coronavirus TWC,gi¦33304219¦gb¦AY351680.1¦ SARS coronavirus ZMY 1,gi¦31416305¦gb¦AY278490.3° SARS coronavirus BJ03,gi¦30910859¦gb¦AY297028.1¦ SARS coronavirus ZJ01,gi¦30421451¦gb¦AY282752.1¦ SARS coronavirus CUHK-Su10, SARS coronavirusSZ16, gi¦34482137¦gb¦AY304486.1¦ SARS coronavirus SZ3gi¦30027610¦gb¦AY278554.2¦ SARS coronavirus CUHK-W1,gi¦31416306¦gb¦AY279354.2¦ SARS coronavirus BJ04,gi¦37576845¦gb¦AY427439.1¦SARS coronavirus AS,gi¦37361915¦gb¦AY283798.2¦ SARS coronavirus Sin2774,gi¦31416290¦gb¦AY278489.2¦ SARS coronavirus GD01,gi¦30468042¦gb¦AY283794.1¦ SARS coronavirus Sin2500,gi¦30468043¦gb¦AY283795.1¦ SARS coronavirus Sin2677,gi¦30468044¦gb¦AY283796.1¦ SARS coronavirus Sin2679,gi¦30468045¦gb¦AY283797.1¦ SARS coronavirus Sin2748,gi¦31982987¦gb¦AY286320.2¦ SARS coronavirus isolate ZJ-HZ01, andgi¦30275666¦gb¦AY278488.2¦ SARS coronavirus BJ01.

The terms “Matrix protein,” “Membrane protein,” “membrane glycoprotein,”and “M protein” refer to a protein that makes the shell of the virus andis closely associated with the lipid membrane that is acquired from thehost cell. This protein is also made in abundance in an infected celland there is typically a strong immune response (particularly, antibodyresponse) to M in CoV infections. The Matrix protein is exemplified, butnot limited to, the sequences in FIG. 30, and by those encoded by thegenomic sequences in gi¦31416292¦gb¦AY278487.3¦ SARS coronavirusBJ02,gi¦30248028¦gb¦AY274119.3¦ SARS coronavirus TOR2,gi¦30698326¦gb¦AY291451.1¦ SARS coronavirus TW1,gi¦33115118¦gb¦AY323977.2¦ SARS coronavirus HSR 1,gi¦35396382¦gb¦AY394850.1¦ SARS coronavirus WHU,gi¦33411459¦dbj¦AP006561.1¦ SARS coronavirus TWY,gi¦33411444¦dbj¦AP006560.1 SARS coronavirus TWS,gi¦33411429¦dbj¦AP006559.1, SARS coronavirus TWK,gi¦33411414¦dbj¦AP006558.1¦ SARS coronavirus TWJ,gi¦33411399¦dbj¦AP006557.1¦ SARS coronavirus TWH,gi¦30023963¦gb¦AY278491.2¦ SARS coronavirus HKU-39849,gi¦33578015¦gb¦AY310120.1¦ SARS coronavirus FRA,gi¦33518725¦gb¦AY362699.1¦ SARS coronavirus TWC3,gi¦33518724¦gb¦AY362698.1¦ SARS coronavirus TWC2,gi¦30027617¦gb¦AY278741.1¦ SARS coronavirus Urbani,gi¦31873092¦gb¦AY321118.1¦ SARS coronavirus TWC,gi¦33304219¦gb¦AY351680.1¦ SARS coronavirus ZMY 1,gi¦31416305¦gb¦AY278490.3¦ SARS coronavirus BJ03,gi¦30910859¦gb¦AY297028.1¦ SARS coronavirus ZJ01,gi¦30421451¦gb¦AY282752.1¦ SARS coronavirus CUHK-Su10,gi¦34482146¦gb¦AY304495.1¦ SARS coronavirus GZ50,gi¦34482139¦gb¦AY304488.1¦ SARS coronavirus SZ16,gi¦34482137¦gb¦AY304486.1¦ SARS coronavirus SZ3,gi¦30027610¦gb¦AY278554.2¦ SARS coronavirus CUHK-W1,gi¦31416306¦gb¦AY279354.2¦ SARS coronavirus BJ04,gi¦37576845¦gb¦AY427439.1¦ SARS coronavirus AS,gi¦37361915¦gb¦AY283798.2¦ SARS coronavirus Sin2774,gi¦31416290¦gb¦AY278489.2¦ SARS coronavirus GD01,gi¦30468042¦gb¦AY283794.1¦ SARS coronavirus Sin2500,gi¦30468043¦gb¦AY283795.1¦ SARS coronavirus Sin2677,gi¦30468044¦gb¦AY283796.1¦ SARS coronavirus Sin2679,gi¦30468045¦gb¦AY283797.1¦ SARS coronavirus Sin2748,gi¦31982987¦gb¦AY286320.2¦ SARS coronavirus isolate ZJ-HZ01, andgi¦30275666¦gb¦AY278488.2¦ SARS coronavirus BJ01.

The terms “E protein,” “envelope protein,” and “small envelope Eprotein” refer to a protein that interacts with the M protein, and thatit is believed to aid in particle formation. It is present at low levelsin the virus particle and at higher levels in an infected cell. The Eprotein is exemplified, but not limited to, the sequences in FIG. 31,and by those encoded by the genomic sequences ingi¦31416292¦gb¦AY278487.3¦ SARS coronavirusBJ02,gi¦30248028¦gb¦AY274119.3¦ SARS coronavirus TOR2,gi¦30698326¦gb¦AY291451.1¦ SARS coronavirus TW1,gi¦33115118¦gb¦AY323977.2¦ SARS coronavirus HSR 1,gi¦35396382¦gb¦AY394850.1¦ SARS coronavirus WHU,gi¦33411459¦dbj¦AP006561.1¦ SARS coronavirus TWY,gi¦33411444¦dbj¦AP006560.1¦ SARS coronavirus TWS,gi¦33411429¦dbj¦AP006559.1¦ SARS coronavirus TWK,gi¦33411414¦dbj¦AP006558.1¦ SARS coronavirus TWJ,gi¦33411399¦dbj¦AP006557.1¦ SARS coronavirus TWH,gi¦30023963¦gb¦AY278491.2¦ SARS coronavirus HKU-39849,gi¦33578015¦gb¦AY310120.1¦ SARS coronavirus FRA,gi¦33518725¦gb¦AY362699.1¦ SARS coronavirus TWC3,gi¦33518724¦gb¦AY362698.1¦ SARS coronavirus TWC2,gi¦30027617¦gb¦AY278741.1¦ SARS coronavirus Urbani,gi¦31873092¦gb¦AY321118¦ SARS coronavirus TWC,gi¦33304219¦gb¦AY351680.1¦ SARS coronavirus ZMY 1,gi¦31416305¦gb¦AY278490.3¦ SARS coronavirus BJ03,gi¦30910859¦gb¦AY297028.1¦ SARS coronavirus ZJ01,gi¦30421451¦gb¦AY282752.1¦ SARS coronavirus CUHK-Su10,gi¦34482146¦gb¦AY304495.1¦ SARS coronavirus GZ50,gi¦34482139¦gb¦AY304488.1¦ SARS coronavirus SZ16,gi¦34482137¦gb¦AY304486.1¦ SARS coronavirus SZ3,gi¦30027610¦gb¦AY278554.2¦ SARS coronavirus CUHK-W1,gi¦31416306¦gb¦AY279354.2¦ SARS coronavirus BJ04,gi¦37576845¦gb¦AY427439.1¦ SARS coronavirus AS,gi¦37361915¦gb¦AY283798.2¦ SARS coronavirus Sin2774,gi¦31416290¦gb¦AY278489.2° SARS coronavirus GD01,gi¦30468042¦gb¦AY283794.1¦ SARS coronavirus Sin2500,gi¦30468043¦gb¦AY283795.1¦ SARS coronavirus Sin2677,gi¦30468044¦gb¦AY283796.1¦ SARS coronavirus Sin2679,gi¦30468045¦gb¦AY283797.1¦ SARS coronavirus Sin2748,gi¦31982987¦gb¦AY286320.2¦ SARS coronavirus isolate ZJ-HZ01, andgi¦30275666¦gb¦AY278488.2¦ SARS coronavirus BJ01.

The terms “Replicase protein,” “polyprotein 1a,” “polypeptide 1a,”“polypeptide 1b,” “polyprotein 1b,” “polyprotein 1ab” and “Pol 1a/b”refer to a relatively large polyprotein that is produced upon infectionof a cell by coronaviruses, and that encodes proteins required forgenome replication. The polyprotein is encoded by about the 5′ twothirds of the genome and it is produced very early during a CoVinfection. The polyprotein is autocatalytically cleaved by encodedproteases (e.g., 3C-like protease) into many proteins that are presentin an infected cell but are not packaged in the virus particle. Theseinclude, without limitation, the RNA dependent RNA polymerase, ahelicase and proteases (e.g., 3C-like, P11 and P12). The polyprotein 1a,1b and 1ab are exemplified, but not limited to, the sequences in FIGS.32-39, and by those encoded by the genomic sequences ingi¦31416292¦gb¦AY278487.3¦ SARS coronavirus BJ02,gi¦30248028¦gb¦AY274119.3¦ SARS coronavirus TOR2,gi¦30698326¦gb¦AY291451.1¦ SARS coronavirus TW1,gi¦33115118¦gb¦AY323977.2¦ SARS coronavirus HSR 1,gi¦35396382¦gb¦AY394850.1¦ SARS coronavirus WHU,gi¦33411459¦dbj¦AP006561.1¦ SARS coronavirus TWY,gi¦33411444¦dbj¦AP006560.1¦ SARS coronavirus TWS,gi¦33411429¦dbj¦AP006559.1¦ SARS coronavirus TWK,gi¦33411414¦dbj¦AP006558.1¦ SARS coronavirus TWJ,gi¦33411399¦dbj¦AP006557.1¦ SARS coronavirus TWH,gi¦30023963¦gb¦AY278491.2¦ SARS coronavirus HKU-39849,gi¦33578015¦gb¦AY310120.1¦ SARS coronavirus FRA,gi¦33518725¦gb¦AY362699.1¦ SARS coronavirus TWC3,gi¦33518724¦gb¦AY362698.1¦ SARS coronavirus TWC2,gi¦30027617¦gb¦AY278741.1° SARS coronavirus Urbani,gi¦31873092¦gb¦AY321118.1¦ SARS coronavirus TWC,gi¦33304219¦gb¦AY351680.1¦ SARS coronavirus ZMY 1,gi¦31416305¦gb¦AY278490.3¦ SARS coronavirus BJ03,gi¦30910859¦gb¦AY297028.1¦ SARS coronavirus ZJ01,gi¦30421451¦gb¦AY282752.1¦ SARS coronavirus CUHK-Su10,gi¦34482146¦gb¦AY304495.1¦ SARS coronavirus GZ50,gi¦34482139¦gb¦AY304488.1¦ SARS coronavirus SZ16,gi¦34482137¦gb¦AY304486.1¦ SARS coronavirus SZ3,gi¦30027610¦gb¦AY278554.2¦ SARS coronavirus CUHK-W1,gi¦31416306¦gb¦AY279354.2¦ SARS coronavirus BJ04,gi¦37576845¦gb¦AY427439.1¦ SARS coronavirus AS,gi¦37361915¦gb¦AY283798.2¦ SARS coronavirus Sin2774,gi¦31416290¦gb¦AY278489.2¦ SARS coronavirus GD01,gi¦30468042¦gb¦AY283794.1¦ SARS coronavirus Sin2500,gi¦30468043¦gb¦AY283795.1¦ SARS coronavirus Sin2677,gi¦30468044¦gb¦AY283796.1¦ SARS coronavirus Sin2679,gi¦30468045¦gb¦AY283797.1¦ SARS coronavirus Sin2748,gi¦31982987¦gb¦AY286320.2¦ SARS coronavirus isolate ZJ-HZ01, andgi¦30275666¦gb¦AY278488.2¦ SARS coronavirus BJ01.

As disclosed herein, in one preferred embodiment, the sgRNA comprises aleader sequence operably linked to the amino terminal region of theSpike protein. This sequence may be amplified by RT-PCR using theprimers SARS-1 (5′-ATATTAGGTTTTTACCTACCCAGG-3′) (SEQ ID NO:69) whichbinds to the leader sequence from nucleotides 1-24 and primerSARS-21,593R (5′-AGTATGTTGAGTGTAATTAGGAG-3′) (SEQ ID NO:70) which bindsto nucleotides encoding the Spike glycoprotein.

ii. gRNA

The invention's methods may further comprises detecting SARS-coronavirusgRNA. The terms “genomic RNA” and “gRNA” are used interchangeably torefer to at least a portion of the genomic sequence such as thatexemplified in by the genome sequences of SARS coronavirus Urbani(GenBank accession # AY278741, FIG. 7), SARS coronavirus Tor2 (GenBankaccession # AY274119, FIG. 8), SARS coronavirus CUHK-W1 (GenBankaccession # AY278554, FIG. 9), SARS-CoV Shanhgai LY (GenBank accession #H012999, FIGS. 10-13; GenBank accession # AY322205, FIG. 20; GenBankaccession # AY322206, FIG. 21), SARS-CoV Shanghai QXC (GenBank accession# AH013000, FIGS. 14-16; GenBank accession # AY322208, FIG. 17; GenBankaccession # AY322197, FIG. 18; GenBank accession # AY322199, FIG. 19),and SARS-CoV ZJ-HZ01 (GenBank accession # AY322206, FIG. 22),gi¦31416292¦gb¦AY278487.3¦ SARS coronavirusBJ02,gi¦30248028¦gb¦AY274119.3¦ SARS coronavirus TOR2,gi¦30698326¦gb¦AY291451.1¦ SARS coronavirus TW1,gi¦33115118¦gb¦AY323977.2¦ SARS coronavirus HSR 1,gi¦35396382¦gb¦AY394850.1¦ SARS coronavirus WHU,gi¦33411459¦dbj¦AP006561.1¦ SARS coronavirus TWY,gi¦33411444¦dbj¦AP006560.1¦ SARS coronavirus TWS,gi¦33411429¦dbj¦AP006559.1¦ SARS coronavirus TWK,gi¦33411414¦dbj¦AP006558.1¦ SARS coronavirus TWJ,gi¦33411399¦dbj¦AP006557.1¦ SARS coronavirus TWH,gi¦30023963¦gb¦AY278491.2¦ SARS coronavirus HKU-39849,gi¦33578015¦gb¦AY310120.1¦ SARS coronavirus FRA,gi¦33518725¦gb¦AY362699.1¦ SARS coronavirus TWC3,gi¦33518724¦gb¦AY362698.1¦ SARS coronavirus TWC2,gi¦30027617¦gb¦AY278741.1¦ SARS coronavirus Urbani,gi¦31873092¦gb¦AY321118.1¦ SARS coronavirus TWC,gi¦33304219¦gb¦AY351680.1¦ SARS coronavirus ZMY 1,gi¦31416305¦gb¦AY278490.3¦ SARS coronavirus BJ03,gi¦30910859¦gb¦AY297028.1¦ SARS coronavirus ZJ01,gi¦30421451¦gb¦AY282752.1¦ SARS coronavirus CUHK-Su10,gi¦34482146¦gb¦AY304495.1¦ SARS coronavirus GZ50,gi¦34482139¦gb¦AY304488.1¦ SARS coronavirus SZ16,gi¦34482137¦gb¦AY304486.1¦ SARS coronavirus SZ3,gi¦30027610¦gb¦AY278554.2¦ SARS coronavirus CUHK-W1,gi¦31416306¦gb¦AY279354.2¦ SARS coronavirus BJ04,gi¦37576845¦gb¦AY427439.1¦ SARS coronavirus AS,gi¦37361915¦gb¦AY283798.2¦ SARS coronavirus Sin2774,gi¦31416290¦gb¦AY278489.2¦ SARS coronavirus GD01,gi¦30468042¦gb¦AY283794.1¦ SARS coronavirus Sin2500,gi¦30468043¦gb¦AY283795.1¦ SARS coronavirus Sin2677,gi¦30468044¦gb¦AY283796.1¦ SARS coronavirus Sin2679,gi¦30468045¦gb¦AY283797.1¦ SARS coronavirus Sin2748,gi¦31982987¦gb¦AY286320.2¦ SARS coronavirus isolate ZJ-HZ01, andgi¦30275666¦gb¦AY278488.2¦ SARS coronavirus BJ01.

Exemplary genomic RNA includes, without limitation, at least a portionof orf1ab polyprotein, orf1a polyprotein, Spike glycoprotein, Orf3a,Orf3a, Orf4b, Orf6, Orf7a, Orf7b, Orf8A, Orf8b, Nucleocapsid protein,Envelope protein E, and Membrane glycoprotein M.

In one preferred embodiment, the gRNA is at least a portion of thePolyprotein lab (also referred to as Polypeptide lab) gene. In oneembodiment, detection of at least a portion of this gene distinguishesbetween sgRNA and gRNA, while detection 3′ to the polyprotein lab genedetects both gRNa and sgRNA, without distinguishing between them. In oneembodiment, the gRNA is of the polyprotein lab gene nucleotides fromabout 1 to about 21,485 of the Urbani strain (FIG. 7, GenBank accession# AY278741). In another embodiment, the gRNA is of the polyprotein labgene nucleotides from about 250 to about 21470 of the CUHK strain (FIG.9, GenBank accession # AY278554). In a further embodiment, the gRNA isof the polyprotein lab gene nucleotides from about 186 to about 1706(GenBank accession # AH012999, FIG. 11) from about 1 to about 10,546(GenBank accession # AH012999, FIG. 12), from about 186 to about 1,706(GenBank accession # AY322205, FIG. 20), and from about 1 to about10,546 (GenBank accession # AY322206, FIG. 21) of the Shanghai LYstrain. In an alternative embodiment, the gRNA is of the polyprotein labgene nucleotides from about 1 to about 3536 (GenBank accession #AH013000, FIG. 14), from about 1 to about 5262 (GenBank accession #AH013000, FIG. 15), and from about 1 to about 3,536 (GenBank accession #AY322197, FIG. 18) of the Shanghai QXC strain.

In another embodiment, the gRNA is at least a portion of the Polyprotein1b (also referred to as Polyprotein 1b) gene. As disclosed herein, theinventors selected an exemplary sequence (tgctaactacattttctggagg) (SEQID NO:71) in Polypeptide-1b to favor conditions for the exemplarymultiplex RT-PCR reaction.

iii. Detecting Nucleic Acids, Proteins, and Virions

Methods for detecting RNA (such as gRNA and sgRNA) are known in the art,and include, but are not limited to, Northern blot, ribonucleaseprotection assay, and polymerase chain reaction.

In one embodiment, RNA (such as gRNA and sgRNA) is detected by Northernblot. The term “Northern blot” as used herein refers to the analysis ofRNA by electrophoresis of RNA on agarose gels to fractionate the RNAaccording to size followed by transfer of the RNA from the gel to asolid support, such as nitrocellulose or a nylon membrane. Theimmobilized RNA is then probed with a labeled oligo-deoxyribonucleotideprobe or DNA probe to detect RNA species complementary to the probeused. Northern blots provide information on both size and abundance oftarget RNA species. Northern blots are a standard tool of molecularbiologists (J. Sambrook, et al. “Molecular Cloning: A LaboratoryManual,” Third Edition, Publ. Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y.).

In another embodiment, RNA (such as gRNA and sgRNA) is detected byribonuclease protection assay. Ribonuclease protection assays are usedto measure the abundance of specific RNAs and to map their topologicalfeatures. The method involves hybridization of test samples tocomplementary radiolabeled RNA probes (riboprobes), followed bydigestion of non-hybridized sequences with one or moresingle-strand-specific ribonucleases. At the end of the digestion, theribonucleases are inactivated, and the protected fragments ofradiolabeled RNA are analyzed by polyacrylamide gel electrophoresis andautoradiography. The ribonuclease protection assay is more sensitivethan the northern blot. The method can detect several target speciessimultaneously, and because the intensity of the signal is directlyproportional to the concentration of target RNA, comparisons of thelevel of expression of the target gene in different tissues can beaccomplished. Methods for ribonuclease protection assay are standard inthe art (J. Sambrook, et al., supra).

In a further embodiment, RNA (such as gRNA and sgRNA) is detected byamplification of a target RNA sequence using reverse transcriptasepolymerase chain reaction. The term “amplification” is defined as theproduction of additional copies of a nucleic acid sequence. The terms“reverse transcription polymerase chain reaction” and “RT-PCR” refer toa method for reverse transcription of an RNA sequence to generate amixture of cDNA sequences, followed by increasing the concentration of adesired segment of the transcribed cDNA sequences in the mixture withoutcloning or purification. Typically, RNA is reverse transcribed using aone or two primers prior to PCR amplification of the desired segment ofthe transcribed DNA using two primers.

Polymerase chain reaction technologies are well known in the art(Dieffenbach C W and G S Dveksler (1995) PCR Primer, a LaboratoryManual, Cold Spring Harbor Press, Plainview N.Y.). PCR describes amethod for increasing the concentration of a segment of a targetsequence in a mixture of DNA without cloning or purification. Thisprocess for amplifying the target sequence consists of introducing alarge excess of two oligonucleotide primers to the DNA mixturecontaining the desired target sequence, followed by a precise sequenceof thermal cycling in the presence of a DNA polymerase. The two primersare complementary to their respective strands of the double strandedtarget sequence. To effect amplification, the mixture is denatured andthe primers then annealed to their complementary sequences within thetarget molecule. Following annealing, the primers are extended with apolymerase so as to form a new pair of complementary strands. The stepsof denaturation, primer annealing and polymerase extension can berepeated many times (i.e., denaturation, annealing and extensionconstitute one “cycle”; there can be numerous “cycles”) to obtain a highconcentration of an amplified segment of the desired target sequence.The length of the amplified segment of the desired target sequence isdetermined by the relative positions of the primers with respect to eachother, and therefore, this length is a controllable parameter. By virtueof the repeating aspect of the process, the method is referred to as the“polymerase chain reaction” (hereinafter “PCR”). Because the desiredamplified segments of the target sequence become the predominantsequences (in terms of concentration) in the mixture, they are said tobe “PCR amplified.”

With PCR, it is possible to amplify a single copy of a specific targetsequence in genomic DNA to a level detectable by several differentmethodologies (e.g., hybridization with a labeled probe; incorporationof biotinylated primers followed by avidin-enzyme conjugate detection;and/or incorporation of ³²P-labeled deoxyribonucleotide triphosphates,such as dCTP or dATP, into the amplified segment).

As used herein, the term “primer” refers to an oligonucleotide, whetheroccurring naturally as in a purified restriction digest or producedsynthetically, which is capable of acting as a point of initiation ofsynthesis when placed under conditions in which synthesis of a primerextension product which is complementary to a nucleic acid strand isinduced, (i.e., in the presence of nucleotides and an inducing agentsuch as DNA polymerase and at a suitable temperature and pH). The primeris preferably single stranded for maximum efficiency in amplification,but may alternatively be double stranded. If double stranded, the primeris first treated to separate its strands before being used to prepareextension products. Preferably, the primer is anoligodeoxyribonucleotide. The primer is selected such that it issufficiently long to prime the synthesis of extension products in thepresence of the inducing agent. Suitable lengths of the primers may beempirically determined and depend on factors such as temperature, sourceof primer and the use of the method. In one embodiment, the primers maybe from 3 to 100, preferably from 3 to 50, more preferably from 3 to 25nucleotide bases in length.

As used herein, the terms “PCR product” and “amplification product”refer to the resultant mixture of compounds after two or more cycles ofthe PCR steps of denaturation, annealing and extension are complete.These terms encompass the case where there has been amplification of oneor more segments of one or more target sequences.

In one embodiment of the invention using RT-PCR, the inventors designedoligonucleotide RT-PCR primers that will amplify genomic RNA or thesgRNA that is specific to the leader-body junction and virusreplication. The inventors probed for sgRNA since the presence ofgenomic RNA alone could result from residual input virus, while thepresence of newly synthesized subgenomic RNA is indicative of virusentry and replication initiation. Genomic RNA was detected by amplifyinga region between the 1b coding region and the sequence encoding theSpike (S) glycoprotein. Subgenomic RNA was detected using a primerspecific to the leader sequence in conjunction with the reverse primerin S that is used for the genomic RNA detection. This procedure could bemodified for any sgRNA and sensitivity could be increased by utilizing3′ genes (e.g. Nucleocapsid). However, the inventors decided to useprimers specific for S because it was their opinion that this geneclearly differentiates genomic and subgenomic RNA molecules and itdecreases false positives that result from viral sgRNA packaging. TheSARS-CoV primer sets were multiplexed with primers for glyceraldehyde 3′phosphate dehydrogenase (G3PDH). These primers were designed to amplifyG3PDH from multiple species to serve as a positive control for RNAintegrity and cDNA production. A one step RT-PCR procedure (Qiagen) waschosen to increase sensitivity over two step procedures because both“forward” and “reverse” primers can serve as reverse transcriptionprimers of antisense and sense coronavirus RNAs, respectively. Thereaction conditions (temperatures, MgCl concentrations etc.) for themultiplexed assay were optimized using SARS-CoV infected Vero E6 cells.

The sensitivity of an exemplary RT-PCR assay of SARS-CoV sgRNA wasdetermined by analyzing RNA isolated from Vero E6 cells inoculated withserial 10-fold dilutions of SARS-CoV. Vero E6 cells were inoculated withinput multiplicities of infection (MOI) ranging from 10⁻¹ to 10⁻⁹ orwere mock inoculated. Total RNA was isolated and subjected to multiplexRT-PCR at 1 h and 24 h post inoculation (FIG. 2). Input genomic RNA wasdetected at 1 PFU per 10,000 cells (FIG. 2, 1 hour panel). Newlysynthesized gRNA and sgRNA was detectable at 1 PFU/million cells (FIG.2, 24 hour panel).

In any of the methods of the invention that employ detection of SARS-CoVgRNA and/or sgRNA, it may be desirable to use a negative control. Dataherein shows that exemplarynegative control cells include baby hamsterkidney cells (BHK-21) (FIG. 2A), MRC-5, MDCK, AK-D, L2, and HRT-18 cells(FIG. 3) which did not produce either gRNA or sgRNA following infectionwith SARS-CoV.

In another embodiment, the invention's methods may employ detecting oneor more SARS-coronavirus polypeptide (such as an antigen). Thepolypeptides may be detected by methods known in the art, such asWestern blot. The terms “Western blot,” “Western immunoblot,”“immunoblot,” and “Western” refer to the immunological analysis ofprotein(s), polypeptides or peptides that have been immobilized onto amembrane support. The proteins are first resolved by acrylamide gelelectrophoresis to separate the proteins, followed by transfer of theprotein from the gel to a solid support, such as nitrocellulose or anylon membrane. The immobilized proteins are then exposed to an antibodyhaving reactivity towards an antigen of interest. The binding of theantibody (i.e., the primary antibody) is detected by use of a secondaryantibody which specifically binds the primary antibody. The secondaryantibody is typically conjugated to an enzyme which permitsvisualization by the production of a colored reaction product orcatalyzes a luminescent enzymatic reaction (e.g., ECL reagent,Amersham). The SARS-CoV polypeptides (such as antigens) may also bedetected using enzyme-linked immunosorbent assay (ELISA), enzyme-basedhistochemical assays, using fluorescent, radioactive, and/or luminescentsystems.

In yet another embodiment, the invention's methods employ detecting theproduction of SARS-coronavirus virions directly or indirectly by using,for example, electron microscopy, CPE, and infection of cells (asdisclosed herein).

G. Detecting Replication of SARS-CoV Using the Invention's ExemplaryCells

The invention provides methods for detecting the presence ofSARS-coronavirus in a sample, comprising: a) providing: (i) a sample;and (ii) cells chosen from one or more of the exemplary HEK-293T, Huh-7,Mv1Lu, pRHMK and pCMK; b) inoculating the cells with the sample toproduce inoculated cells; and c) detecting the presence of theSARS-coronavirus in the inoculated cells. These methods are useful in,for example, diagnosing the presence of SARS-CoV in samples, screeningagents for their activity in reducing SARS-CoV infection, determiningthe relative efficacy of agents and/or modalities of treatment inaltering (e.g., increasing or reducing) the levels SARS infection.

i. Cultures Containing the Invention's Cells

In one embodiment, any of the invention's methods may be performed usingsingle cell type culture. The term “single-cell type culture” refers toa composition, whether liquid, gel, or solid, which contains one celltype (for example, HEK-293T alone, Huh-7 alone, Mv1Lu alone, pRHMKalone, or pCMK alone).

The invention further employs mixed cell type cultures. As used herein,the term “mixed-cell type culture” refers to a composition, whetherliquid, gel, or solid, which contains a mixture of two or more types ofcells wherein the cell types are mingled together. For example, amixed-cell type culture may contain cells from different tissues ororgans from the same species and same genus. Alternatively, a mixed-celltype culture may contain cells from different species in the same genus.Yet another alternative is that a mixed-cell type culture contain cellsfrom a different genus. The present invention encompasses anycombination of cell types. Such combinations may be suitable in, forexample, the detection, identification, and/or quantitation of virusesin samples, including mixed cell cultures in which all of the cell typesused are not genetically engineered, mixtures in which one or more ofthe cell types are genetically engineered and the remaining cell typesare not genetically engineered, and mixtures in which all of the celltypes are genetically engineered.

The term “cell type different from a specifically identified cell type”as used herein means any cell type that differs in any way from thespecifically identified cell type. This term includes, withoutlimitation, the parental cells from which the specifically identifiedcell type has been established (e.g., by serial culture, transfectionwith one or more nucleotide sequences of interest, immortalization,etc.).

An advantage of using one or more of the invention's cells (such as theexemplary HEK-293T, Huh-7, Mv1Lu, pRHMK and/or pCMK cell, etc.) inmixed-cell-type culture with each other is that they may providedifferent SARS-CoV antigens that may be used for vaccine and/or antibodyproduction. An advantage of using one or more of the invention's cells(such as the exemplary HEK-293T, Huh-7, Mv1Lu, pRHMK and/or pCMK cell,etc.) in mixed-cell-type culture with other cell types, is that suchcultures provide rapid and sensitive assay systems in a single unit forthe detection of multiple viruses, and they also eliminate the need formultiple cell lines cultured in individual containers.

In one embodiment, the mixed cell type culture contains one or more ofthe exemplary HEK-293T, Huh-7, Mv1Lu, pRHMK and pCMK cell together. Inanother embodiment, the mixed cell type culture contains one or more ofthe exemplary HEK-293T, Huh-7, Mv1Lu, pRHMK and pCMK cell together, aswell as other cell types. These mixed cell type cultures are useful in,for example, detecting the presence of viruses other than SARS-CoV. Forexample, mixed cell type cultures containing Mv1Lu cells and A549 cells(ATCC No. CCL185) may be used for detection of SARS-CoV, parainfluenzaviruses, and influenza viruses by Mv1Lu cells, as well as detection ofHerpes viruses, enteroviruses, adenoviruses, myxoviruses, andparamyviruses by A549 cells. Mixed cell cultures of Mv1Lu and A549 areknown in the art (sold as “R-MIX™” By Diagnostic Hybrids Inc., Ohio)(U.S. Pat. No. 6,376,172, incorporated by reference in its entirety).

While not limiting the invention to any particular cell type, exemplarycell lines which may be used in mixed-cell type cultures with each otherand/or with any one or more of the invention's cells (such as theexemplary HEK-293T, Huh-7, Mv1Lu, pRHMK and/or pCMK cell, etc.) arelisted in Table 2. TABLE 2 Exemplary Cell Lines For Mixed-Cell TypeCultures With The Invention's Cells Cell Line ATCC No. SourceVirus^((a)) primary none^((b)) Kidney, rhesus monkey Herpes, entero,monkey adeno, myxo, paramy BS-C-1 CCL26 Kidney, African green monkeyHerpes, entero, adeno, myxo, paramy CV-1 CCL70 Kidney, African greenmonkey Herpes, entero, adeno, myxo, paramy Vero CCL81 Kidney, Africangreen monkey Herpes, entero, adeno, myxo, paramy Vero 76 CRL1587 Kidney,African green monkey Herpes, entero, adeno, myxo, paramy Vero C1008CRL1586 Kidney, African green monkey Herpes, entero, adeno, myxo, paramyVero 76 CCL81 Kidney, African green monkey Herpes, entero, adeno, myxo,paramy Cos-1 CRL1650 Kidney, African green monkey, Herpes, entero,transformed adeno, myxo, paramy Cos-7 CRL1651 Kidney, African greenmonkey, Herpes, entero, transformed adeno, myxo, paramy FRhK-4 CRL1688Kidney, fetal rhesus monkey Herpes, entero, adeno, myxo, paramy LLC-MK2CCL7 Kidney, rhesus monkey Herpes, entero, original adeno, myxo, paramyLLC-MK2 CCL7.1 Kidney, rhesus monkey Herpes, entero, derivative adeno,myxo, paramy MDCK CCL34 Kidney, canine Herpes, entero, adeno, myxo,paramy CCD-13 Lu CCL200 Lung, human Herpes, entero, adeno, paramy CCD-8Lu CCL201 Lung, human Herpes, entero, adeno, paramy CCD-14 Br CCL203Bronchiole, human Herpes, entero, adeno, myxo, paramy CCD-16 Lu CCL204Lung, human Herpes, entero, adeno, paramy CCD-18 Lu CCL205 Lung, humanHerpes, entero, adeno, paramy CCD-19 Lu CCL210 Lung, human Herpes,entero, adeno, paramy Hs888 Lu CCL211 Lung, human Herpes, entero, adeno,paramy MRC-9 CCL212 Lung, human Herpes, entero, adeno, paramy CCD-25 LuCCL215 Lung, human Herpes, entero, adeno, paramy WiDr CCL218 Colon,adenocarcinoma, human Herpes, entero, adeno DLD-1 CCL221 Colon,adenocarcinoma, human Herpes, entero, adeno COLO205 CCL222 Colon,adenocarcinoma, human Herpes, entero, adeno HCT-15 CCL222 Colon,adenocarcinoma, human Herpes, entero, adeno SW 480 CCL228 Colon,adenocarcinoma, human Herpes, entero, adeno LOVO CCL229 Colon,adenocarcinoma, human Herpes, entero, adeno SW403 CCL230 Colon,adenocarcinoma, human Herpes, entero, adeno SW48 CCL231 Colon,adenocarcinoma, human Herpes, entero, adeno SW116 CCL233 Colon,adenocarcinoma, human Herpes, entero, adeno SW1463 CCL234 Colon,adenocarcinoma, human Herpes, entero, adeno SW837 CCL235 Rectum,adenocarcinoma, human Herpes, entero, adeno SW948 CCL237 Colon,adenocarcinoma, human Herpes, entero, adeno SW1417 CCL238 Colon,adenocarcinoma, human Herpes, entero, adeno FHs74 Int CCL241 Smallintestine, adenocarcinoma, Herpes, entero, adeno human HCT-8 CCL244Adenocarcinoma, ileococal Herpes, entero, adeno HCT-116 CCL247 Coloncarcinoma, human Herpes, entero, adeno T84 CCL248 Colon carcinoma, humanHerpes, entero, adeno NCI-H747 CCL252 Cecum, adenocarcinoma, humanHerpes, entero, adeno NCI-H508 CCL253 Cecum, adenocarcinoma, humanHerpes, entero, adeno LS123 CCL255 Colon, human, adenocarcinoma Herpes,entero, adeno CaCo-2 HTB37 Colon, adenocarcinoma, human Herpes, entero,adeno HT-29 HTB38 Colon, adenocarcinoma, human Herpes, entero, adenoSK-CO-1 HTB39 Colon, adenocarcinoma, human Herpes, entero, adeno HuTu 80HTB40 Duodenum, adenocarcinoma, human Herpes, entero, adeno A253 HTB41Epidemoid carcinoma Herpes, entero, adeno, paramyo A704 HTB45 Kidneyadenocarcinoma, human Herpes, entero, adeno, paramyo Hela CCL2Epitheloid carcinoma, cervix, Herpes, entero, human adeno, myxo, paramyHela CCL2.1 Epitheloid carcinoma, cervix, Herpes, entero, human adeno,myxo, paramy Hela53 CCL2.2 Epitheloid carcinoma, cervix, Herpes, entero,human adeno, myxo, paramy L-132 CCL5 Embryonic lung, human, Hela Herpes,entero, marker adeno, myxo, paramy Intestine CCL6 Embryonic intestine,human, Herpes, entero, adeno Hela marker BHK-21 CCL10 Kidney, synisteror golden Herpes, entero, hamster adeno, myxo, paramy Hak CCL15 Kidney,syn hamster Herpes, entero, adeno, myxo, paramy KB CCL17 Epidermoidcarcinoma oral, human Herpes, entero, adeno, paramy Hep-2 CCL23Epidermoid carcinoma larynx, Herpes, entero, adeno, human paramy WishCCL25 Ammion, human Herpes, entero, adeno Detroit 532 CCL54 Skin, humanHerpes, entero, adeno FL CCL62 Ammion, human Herpes, entero Detroit 525CCL65 Skin, human Herpes, entero, adeno Detroit 529 CCL66 Skin, humanHerpes, entero, adeno Detroit 510 CCL72 Skin, human Herpes, entero,adeno WI-38 CCL75 Lung, diploid human Herpes, entero, adeno, paramyWI-38 VA13 CCL75.1 Lung, diploid human, SV-40 Herpes, entero, adeno,transformed paramy Citrullinemia CCL76 Skin, human Herpes, entero,adeno, paramy Spik (NBL-10) CCL78 Kidney, dolphin Herpes, entero, adenoDetroit 539 CCL84 Skin, human Herpes, entero, adeno Cridu Chat CCL90Skin, human Herpes, entero, adeno WI26 VA4 CCL95.1 Lung, human Herpes,entero, adeno, paramy BeWo CCL98 Choriocarcinoma, human Herpes, entero,adeno SW-13 CCL105 Adenocarcinoma, human, Herpes, entero, adeno adrenalcortex Detroit 548 CCL116 Skin Herpes, entero, adeno Detroit 573 CCL117Skin Herpes, entero, adeno HT-1080 CCL121 Fibrocarcinoma, human Herpes,entero, adeno HG 261 CCL122 Skin, human Herpes, entero, adeno C211CCL123 Skin, human Herpes, entero, adeno Amdur II CCL124 Skin, humanHerpes, entero, adeno CHP 3 (M.W.) CCL132 Skin, human, fibroid likeHerpes, entero, adeno CHP 4 (W.W.) CCL133 Skin, human, fibroid likeHerpes, entero, adeno RD CCL136 Rhabdomyosarcoma Herpes, entero, adenoHEL 299 CCL137 Lung, diploid Herpes, entero, adeno, paramy Detroit 562CCL138 Carcinoma, pharynx Herpes, entero, adeno, myxo, paramy MRC-5CCL171 Lung, diploid, human Herpes, entero, adeno, paramy A-549 CCL185Lung, carcinoma, human Herpes, entero, adeno, myxo, paramy IMR-90 CCL186Lung, carcinoma, human Herpes, entero, adeno, myxo, paramy LS180 CCL187Colon, adenocarcinoma, human Herpes, entero, adeno LS174T CCL188 Colon,adenocarcinoma, human Herpes, entero, adeno NCI-H292 CCL-1848Mucoepidermoid, human Respir. syncytial virus BHK/ICP6La CCL-12072 cZ-5CV-1 CCL-70 hs27 HFF; CRL-1634 Mv1Lu CCL-64 McCoy CCL-1696 MRC-5 CCL-171Vero CCL-81 MDCK (NBL-2) CCL-34 BHK21 CCL-10 Mv1Lu-hF PTA-4737 Lung,epithelial, mink Influenza, parainfluenza^((a))Herpes = Herpes virusesEntero = EnterovirusesAdeno = AdenovirusesMyxo = MyxovirusesParamy = Paramyxoviruses^((b))Primary monkey kidney cells may be obtained from DiagnosticHybrids (catalog numbers 490102A for shell format and 49-0600A for tubeformat)

In one embodiment, it may be desirable use Mv1Lu cells for thereplication and/or detection of parainfluenza and influenza viruseswithout replication and/or detection of SARS-CoV. This is advantageousin laboratories that diagnose infection with parainfluenza and influenzaviruses, and that do not have access to containment facilities that arerequired for manipulation of SARS-CoV. In one embodiment, this goal maybe achieved by incubating a test sample with Mv1Lu cells for up to 24hours. This is based on data herein (FIG. 4B) which shows that SARS-CoVwas not produced by Mv1Lu cells within 24 hours p.i. In anotherembodiment, this goal may be achieved by contacting one or more of theMv1Lu cells and the sample with antibody specific for one or moreSARS-coronavirus antigen.

In a further embodiment, the goal of reducing infection of Mv1Lu cellsby SARS-coronavirus, while not substantially reducing susceptibility ofMv1Lu cells to parainfluenza and/or influenza viruses, may be attainedby contacting the Mv1Lu cells and/or sample that is being tested with aprotease inhibitor, as further described below.

In another embodiment, it may be desirable to use Mv1Lu cells for thedetection and/or proliferating of parainfluenza and influenza viruses inaddition to detection and/or replication of SARS-CoV, such as where thespecificity of action of certain reagents on different viruses is beinginvestigated. This may be achieved by incubating Mv1Lu cells with a testsample for more than 24 hours (FIG. 4B).

ii. Cells Frozen In Situ

In one embodiment, the invention's cells (such as the exemplaryHEK-293T, Huh-7, Mv1Lu, pRHMK and/or pCMK cell, etc.) are frozen insitu. Methods for the in situ growth, freezing and testing of culturedcells are know in the art (U.S. Pat. No. 6,472,206, incorporated byreference in its entirety). In one embodiment, the in situ frozen cellsare in single cell type culture. In another embodiment, the in situfrozen cells are in mixed cell type culture.

iii. Samples

The invention contemplates contacting the invention's cells (such as theexemplary HEK-293T, Huh-7, Mv1Lu, pRHMK and/or pCMK cell, etc.) with asample for the purpose of, for example and without limitation, detectingand/or quantitating SARS-CoV polypeptides, proteins, and/or virusparticles in a sample. The terms “sample” and “specimen” as used hereinare used in their broadest sense to include any composition that isobtained and/or derived from biological or environmental source, as wellas sampling devices (e.g., swabs) which are brought into contact withbiological or environmental samples. “Biological samples” include thoseobtained from an animal (including humans, domestic animals, as well asferal or wild animals, such as ungulates, bear, fish, lagamorphs,rodents, etc.), body fluids such as urine, blood, plasma, fecal matter,cerebrospinal fluid (CSF), semen, sputum, and saliva, as well as solidtissue. Biological samples also include a cell (such as cell lines,cells isolated from tissue whether or not the isolated cells arecultured after isolation from tissue, fixed cells such as cells fixedfor histological and/or immunohistochemical analysis), tissue (such asbiopsy material), cell extract, tissue extract, and nucleic acid (e.g.,DNA and RNA) isolated from a cell and/or tissue, and the like. Alsoincluded are materials obtained from food products and food ingredientssuch as dairy items, vegetables, meat, meat by-products, and waste.Environmental samples” include environmental material such as surfacematter, soil, water, and industrial materials, as well as materialobtained from food and dairy processing instruments, apparatus,equipment, disposable, and non-disposable items. In one embodiment, thebiological sample is a cell, tissue, and or fluid obtained from amammal, including from the upper respiratory tissues (such asnasopharyngeal wash, nasopharyngeal aspirate, nasopharyngeal swab, andoropharyngeal swab), from the lower respiratory tissues (such asbronchiolar lavage, tracheal aspirate, pleural tap, sputum), blood,plasma, serum, stool, and tissue from any organ such as, withoutlimitation, lung, heart, spleen, liver, brain, kidney, and adrenalglands. These examples are illustrative, and are not to be construed aslimiting the sample types applicable to the present invention.

While not intending to limit the source of the sample, in oneembodiment, the sample is isolated from a mammal. In one embodiment, the“mammal” is rodent (such as mouse and rat, such as cotton rat), primate(including simian and human) ovine, bovine, ruminant, lagomorph,porcine, caprine, equine, canine, feline, avian, etc. Expressly includedare hamster, mink, ferret, pig, cat, and rabbit.

The invention also provides methods for detecting the presence ofSARS-CoV in one or more samples, such as in samples from mammals thathave been treated with anti-SARS-CoV agents. These methods may be usedin, for example, determining the efficacy of a therapeutic modality(such as a chemical drug) in reducing SARS-coronavirus infection in amammal, including a model animal and human. These methods are alsouseful in determining the relative efficacy of different therapeuticmodalities, such as different concentrations of the same drug, the sameconcentration of different drugs, and different combinations of drugs.

Thus, in one embodiment, the invention provides a method for detectingthe presence of SARS-coronavirus in a first sample and in a secondsample, comprising: a) providing: (i) a first sample; (ii) a secondsample; b) contacting test cells chosen from one or more of theinvention's cells (such as the exemplary HEK-293T, Huh-7, Mv1Lu, pRHMKand/or pCMK cell, etc.) with: (i) the first sample to produce a firsttreated sample; and (ii) the second sample to produce a second treatedsample; wherein the exposing is under conditions such that the testcells are infected with SARS-coronavirus; c) detecting the presence ofone or more of SARS-coronavirus gRNA and SARS-coronavirus sgRNA, whereinthe detecting indicates the presence of the SARS-coronavirus. In apreferred embodiment, the detecting step comprises detecting one or moreof: i) absence of SARS-coronavirus gRNA in the first treated sample; ii)reduced level of SARS-coronavirus sgRNA in the first treated samplecompared to the level of sgRNA in the second treated sample; and iii)reduced ratio of SARS-coronavirus sgRNA level to SARS-coronavirus gRNAlevel in the first treated sample compared to in the second treatedsample. Detection of any one or more of these phenomena indicates thatthe first sample contains a reduced level of SARS-coronavirus comparedto the second sample.

In one embodiment, the method comprises detecting an absence ofSARS-coronavirus gRNA in the first treated sample. Without limiting theinvention to any particular mechanism, such detection indicates thatSARS-coronavirus has not adsorbed to cells from which the first samplewas obtained.

In another embodiment, the method comprises detecting an absence ofSARS-coronavirus sgRNA in the first treated sample. Without limiting theinvention to any particular mechanism, such detection indicates thatSARS-coronavirus has not replicated in cells from which the first samplewas obtained.

In a preferred embodiment, the method comprises detecting an absence ofSARS-coronavirus gRNA and SARS-coronavirus sgRNA in the first treatedsample. Without limiting the invention to any particular mechanism, suchdetection indicates that SARS-coronavirus has neither adsorbed to norreplicated in cells from which the first sample was obtained.

In one embodiment, the first sample and the second sample are from amammal. In a preferred embodiment, the first sample is from a mammaltreated with an agent and the second sample is from the mammal that isnot treated with the agent. These steps may be used in, for example,identifying an agent as reducing infection with SARS-CoV in a modelanimal or in human clinical trials.

In another embodiment, the first sample is from a mammal treated with afirst concentration of an agent and the second sample is from the mammaltreated with a second concentration of the agent, wherein the first andsecond concentrations are different. These steps may be used in, forexample, comparing the relative efficacy of different concentrations ofthe same agent in reducing infection with SARS-CoV in a model animal orin human clinical trials.

In a further embodiment, the first sample is from a mammal treated witha first agent and the second sample is from the mammal treated with asecond agent wherein the first and second agents are different. Thesesteps may be used in, for example, comparing the relative efficacy ofdifferent agents in reducing infection with SARS-CoV in a model animalor in human clinical trials.

H. Screening Anti-SARS-CoV Agents

In one embodiment, the invention provides a method for identifying atest agent as altering infection of a cell by SARS-coronavirus,comprising: a) providing cells treated with a test agent, wherein thecells are chosen from one or more of HEK-293T, Huh-7, Mv1Lu, pRHMK andpCMK; and b) detecting an altered level of infection of cells treatedwith the test agent compared to a level of infection of the cells nottreated with the test agent, wherein the detecting identifies the testagent as altering infection of a cell by SARS-coronavirus. The alteredlevel of infection may be a reduced level or an increased level.

This method may be used in, for example, screening anti-SARS-coronavirusdrugs. Anti-SARS-coronavirus drugs may be used as prophylactic agentsand/or therapeutic agents in the treatment of SARS-coronavirus.Anti-SARS-coronavirus drugs may also be used to increase the safety ofhandling cells, such as Mv1Lu cells that are used in clinicallaboratories for and that may be susceptible and/or permissive toSARS-coronavirus. For example, with respect to Mv1Lu cells, which areroutinely used in clinical laboratories for screening infection withinfluenza and parainfluenza viruses, and which show low permissivity toSARS-CoV, particularly useful are anti-SARS CoV drugs that reducepermissivity of Mv1Lu cells to SARS, while not substantially reducingsusceptibility and/or permissivity of Mv1Lu cells to influenza virusand/or parainfluenza virus. The invention's methods are also useful indetermining the efficacy of a drug in reducing infection in a modelmammal and in human clinical trials.

In one embodiment, the detecting step may comprise detectingSARS-coronavirus sgRNA, gRNA, polypeptide and/or virion. In anotherembodiment, the detecting step comprises detecting one or more of: i)absence of SARS-coronavirus gRNA in the treated cells; ii) reduced levelof SARS-coronavirus sgRNA in the treated cells compared to the level ofsgRNA in the cells that are not treated with the test agent; and (iii)reduced ratio of SARS-coronavirus sgRNA level to SARS-coronavirus gRNAlevel in the treated cells compared to in the cells that are not treatedwith the test agent; wherein the detecting identifies the test agent asreducing infection of a cell by SARS-coronavirus.

In another embodiment, it may be desirable to compare the efficacy oftwo agents in reducing infection with SARS-coronavirus. This may beachieved by detecting one or more of: i) reduced level ofSARS-coronavirus sgRNA in the cells treated with a second test agentcompared to the level of sgRNA in the cells treated with the test agent;and ii) reduced ratio of SARS-coronavirus sgRNA level toSARS-coronavirus gRNA level in the cells treated with a second testagent compared to the ratio in the cells treated with the test agent,wherein detecting an increased reduction in one or more of the level ofSARS-coronavirus sgRNA and of the ratio of SARS-coronavirus sgRNA levelto SARS-coronavirus gRNA level in the cells treated with the test agentcompared to the cells treated with the second test agent identifies thetest agent as more efficacious than the second test agent in reducinginfection of a cell by SARS-coronavirus.

The “agent” identified by, and/or used by, the invention's methodsrefers to any type of molecule (for example, a peptide, nucleic acid,carbohydrate, lipid, organic, and inorganic molecule, etc.) obtainedfrom any source (for example, plant, animal, and environmental source,etc.), or prepared by any method (for example, purification of naturallyoccurring molecules, chemical synthesis, and genetic engineeringmethods, etc.). The terms “test compound,” “compound,” “agent,” “testagent,” “molecule,” and “test molecule,” as used herein, refer to anychemical entity, pharmaceutical, drug, and the like that can be used totreat or prevent a disease, illness, sickness, or disorder of bodilyfunction. Agents comprise both known and potential therapeuticcompounds. An agent can be determined to be therapeutic by screeningusing the screening methods of the present invention. A “knowntherapeutic compound” refers to a therapeutic compound that has beenshown (e.g., through animal trials or prior experience withadministration to humans) to be effective in such treatment orprevention. In other words, a known therapeutic compound is not limitedto a compound efficacious in the treatment of SARS-coronavirusinfection. Agents are exemplified by, but not limited to, vaccines,antibodies, nucleic acid sequences such as ribozyme sequences, and otheragents as further described herein.

In one embodiment, the agent is an antibody that is specific for one ormore SARS-coronavirus antigens. The terms “antibody” and“immunoglobulin” are interchangeably used to refer to a glycoprotein ora portion thereof (including single chain antibodies), which is evokedin an animal by an immunogen and which demonstrates specificity to theimmunogen, or, more specifically, to one or more epitopes contained inthe immunogen. The term “antibody” includes polyclonal antibodies,monoclonal antibodies, naturally occurring antibodies as well asnon-naturally occurring antibodies, including, for example, single chainantibodies, chimeric, bifunctional and humanized antibodies, as well asantigen-binding fragments thereof, including, for example, Fab, F(ab′)₂,Fab fragments, Fd fragments, and Ev fragments of an antibody, as well asa Fab expression library. It is intended that the term “antibody”encompass any immunoglobulin (e.g., IgG, IgM, IgA, IgE, IgD, etc.)obtained from any source (e.g., humans, rodents, non-human primates,caprines, bovines, equines, ovines, etc.). The term “polyclonalantibody” refers to an immunoglobulin produced from more than a singleclone of plasma cells; in contrast “monoclonal antibody” refers to animmunoglobulin produced from a single clone of plasma cells. Monoclonaland polyclonal antibodies may or may not be purified. For example,polyclonal antibodies contained in crude antiserum may be used in thisunpurified state.

Naturally occurring antibodies may be generated in any speciesincluding, for example, murine, rat, rabbit, hamster, human, and simianspecies using methods known in the art. Non-naturally occurringantibodies can be constructed using solid phase peptide synthesis, canbe produced recombinantly or can be obtained, for example, by screeningcombinatorial libraries consisting of variable heavy chains and variablelight chains as previously described (Huse et al., Science 246:1275-1281(1989)). These and other methods of making, for example, chimeric,humanized, CDR-grafted, single chain, and bifunctional antibodies arewell known to those skilled in the art (Winter and Harris, Immunol.Today 14:243-246 (1993); Ward et al., Nature 341:544-546 (1989); Hilyardet al., Protein Engineering: A practical approach (IRL Press 1992); andBorrabeck, Antibody Engineering, 2d ed. (Oxford University Press 1995).

Those skilled in the art know how to make polyclonal and monoclonalantibodies which are specific to a desirable polypeptide. For theproduction of monoclonal and polyclonal antibodies, various host animalscan be immunized by injection with the peptide corresponding to anymolecule of interest in the present invention, including but not limitedto rabbits, mice, rats, sheep, goats, etc. In one embodiment, thepeptide is conjugated to an immunogenic carrier (e.g., diphtheriatoxoid, bovine serum albumin (BSA), or keyhole limpet hemocyanin (KLH)).Various adjuvants may be used to increase the immunological response,depending on the host species, including but not limited to Freund's(complete and incomplete), mineral gels such as aluminum hydroxide,surface active molecules such as lysolecithin, pluronic polyols,polyanions, peptides, oil emulsions, keyhole limpet hemocyanins,dinitrophenol, and potentially useful human adjuvants such as BCG(Bacille Calmette-Guerin) and Corynebacterium parvum.

For preparation of monoclonal antibodies directed toward molecules ofinterest in the present invention, any technique that provides for theproduction of antibody molecules by continuous cell lines in culture maybe used (See, e.g., Harlow and Lane, Antibodies: A Laboratory Manual,Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.). Theseinclude but are not limited to the hybridoma technique originallydeveloped by Köhler and Milstein (Köhler and Milstein, Nature256:495-497 (1975)), as well as the trioma technique, the human B-cellhybridoma technique (See e.g., Kozbor et al. Immunol. Today 4:72(1983)), and the EBV-hybridoma technique to produce human monoclonalantibodies (Cole et al., in Monoclonal Antibodies and Cancer Therapy,Alan R. Liss, Inc., pp. 77-96 (1985)). In one embodiment of themonoclonal antibodies are of the IgG class.

In additional embodiments of the invention, monoclonal antibodies can beproduced in germ-free animals utilizing technology such as thatdescribed in PCT/U.S. Ser. No. 90/02545. In addition, human antibodiesmay be used and can be obtained by using human hybridomas (Cote et al.,Proc. Natl. Acad. Sci. U.S.A. 80:2026-2030 (1983)) or by transforminghuman B cells with EBV virus in vitro (Cole et al., in MonoclonalAntibodies and Cancer Therapy, Alan R. Liss, pp. 77-96 (1985)).

Furthermore, techniques described for the production of single chainantibodies (See e.g., U.S. Pat. No. 4,946,778; herein incorporated byreference) can be adapted to produce single chain antibodies thatspecifically recognize one or more SARS antigens. An additionalembodiment of the invention utilizes the techniques described for theconstruction of Fab expression libraries (Huse et al., Science246:1275-1281 (1989)) to allow rapid and easy identification ofmonoclonal Fab fragments with the desired specificity for a particularprotein or epitope of interest (e.g., at least a portion of an AUBP ormammalian exosome).

The invention also contemplates humanized antibodies. Humanizedantibodies may be generated using methods known in the art, includingthose described in U.S. Pat. Nos. 5,545,806; 5,569,825 and 5,625,126,the entire contents of which are incorporated by reference. Such methodsinclude, for example, generation of transgenic non-human animals whichcontain human immunoglobulin chain genes and which are capable ofexpressing these genes to produce a repertoire of antibodies of variousisotypes encoded by the human immunoglobulin genes.

According to the invention, techniques described for the production ofsingle chain antibodies (U.S. Pat. No. 4,946,778; herein incorporated byreference) can be adapted to produce specific single chain antibodies asdesired. An additional embodiment of the invention utilizes thetechniques known in the art for the construction of Fab expressionlibraries (Huse et al., Science, 246:1275-1281 (1989)) to allow rapidand easy identification of monoclonal Fab fragments with the desiredspecificity.

Antibody fragments that contain the idiotype (antigen binding region) ofthe antibody molecule can be generated by known techniques. For example,such fragments include but are not limited to: the F(ab′)2 fragment thatcan be produced by pepsin digestion of an antibody molecule; the Fab′fragments that can be generated by reducing the disulfide bridges of anF(ab′)2 fragment, and the Fab fragments that can be generated bytreating an antibody molecule with papain and a reducing agent.

In the production of antibodies, screening for the desired antibody canbe accomplished by techniques known in the art (e.g., radioimmunoassay,ELISA (enzyme-linked immunosorbent assay), “sandwich” immunoassays,immunoradiometric assays, gel diffusion precipitin reactions,immunodiffusion assays, in situ immunoassays (e.g., using colloidalgold, enzyme or radioisotope labels), Western blots, precipitationreactions, agglutination assays (e.g., gel agglutination assays,hemagglutination assays, etc.), complement fixation assays,immunofluorescence assays, protein A assays, and immunoelectrophoresisassays, etc.

In an alternative embodiment, the agent is a nucleic acid sequence. Theterms “nucleic acid sequence” and “nucleotide sequence” as used hereinrefer to two or more nucleotides which are covalently linked to eachother. Included within this definition are oligonucleotides,polynucleotide, and fragments or portions thereof, DNA or RNA of genomicor synthetic origin which may be single- or double-stranded, andrepresent the sense or antisense strand. Nucleic acid sequences whichare particularly useful in the instant invention include, withoutlimitation, antisense sequences and ribozymes.

In one embodiment, the agent that alters the infection bySARS-coronavirus is an antisense nucleic acid sequence which hybridizeswith at least a portion of SARS-coronavirus genomic RNA and/orsubgenomic RNA. Antisense sequences have been successfully used toinhibit the expression of several genes (Markus-Sekura (1988) Anal.Biochem. 172:289-295; Hambor et al. (1988) J. Exp. Med. 168:1237-1245;and patent EP 140 308), including the gene encoding VCAM1, one of theintegrin α4β1 ligands (U.S. Pat. No. 6,252,043, incorporated in itsentirety by reference). The terms “antisense DNA sequence” and“antisense sequence” as used herein interchangeably refer to adeoxyribonucleotide sequence whose sequence of deoxyribonucleotideresidues is in reverse 5′ to 3′ orientation in relation to the sequenceof deoxyribonucleotide residues in a sense strand of a DNA duplex. A“sense strand” of a DNA duplex refers to a strand in a DNA duplex whichis transcribed by a cell in its natural state into a “sense mRNA.” SensemRNA generally is ultimately translated into a polypeptide. Thus, an“antisense DNA sequence” is a sequence which has the same sequence asthe non-coding strand in a DNA duplex, and which encodes an “antisenseRNA” (i.e., a ribonucleotide sequence whose sequence is complementary toa “sense mRNA” sequence). The designation (−) (i.e., “negative”) issometimes used in reference to the antisense strand, with thedesignation (+) sometimes used in reference to the sense (i.e.,“positive”) strand. Antisense RNA may be produced by any method,including synthesis by splicing an antisense DNA sequence to a promoterwhich permits the synthesis of antisense RNA. The transcribed antisenseRNA strand combines with natural mRNA produced by the cell to formduplexes. These duplexes then block either the further transcription ofthe mRNA or its translation, or promote its degradation.

Antisense oligonucleotide sequences may be synthesized using any of anumber of methods known in the art (such as solid support andcommercially available DNA synthesizers, standard phosphoramidatechemistry techniques, and commercially available services, e.g., Genta,Inc.).

Other molecules which find use as agents for altering infection bySARS-coronavirus include organic molecules, inorganic molecules, andlibraries of any type of molecule, which can be screened using a methodof the invention, and which may be prepared using methods known in theart. These agents are made by methods for preparing oligonucleotidelibraries (Gold et al., U.S. Pat. No. 5,270,163, incorporated byreference); peptide libraries (Koivunen et al. J. Cell Biol., 124:373-380 (1994)); peptidomimetic libraries (Blondelle et al., TrendsAnal. Chem. 14:83-92 (1995)) oligosaccharide libraries (York et al.,Carb. Res. 285:99-128 (1996); Liang et al., Science 274:1520-1522(1996); and Ding et al., Adv. Expt. Med. Biol. 376:261-269 (1995));lipoprotein libraries (de Kruif et al., FEBS Lett., 399:232-236 (1996));glycoprotein or glycolipid libraries (Karaoglu et al., J. Cell Biol.130:567-577 (1995)); or chemical libraries containing, for example,drugs or other pharmaceutical agents (Gordon et al., J. Med. Chem.37:1385-1401 (1994); Ecker and Crook, Bio/Technology 13:351-360 (1995),U.S. Pat. No. 5,760,029, incorporated by reference). Libraries ofdiverse molecules also can be obtained from commercial sources.

I. Administering Anti-SARS-CoV Agents

The invention provides a method for reducing infection bySARS-coronavirus comprising administering a therapeutic amount of anagent to a mammal. The terms “therapeutic amount,” “pharmaceuticallyeffective amount,” “therapeutically effective amount,” “biologicallyeffective amount,” and are used interchangeably herein to refer to anamount which is sufficient to achieve a desired result, whetherquantitative or qualitative. In particular, a pharmaceutically effectiveamount is that amount that results in the reduction, delay, and/orelimination of undesirable effects (such as pathological, clinical,biochemical and the like) in the subject that are associated withinfection with SARS-coronavirus. As used herein, the actual amountencompassed by the term “therapeutic amount” will depend on the route ofadministration, the type of subject being treated, and the physicalcharacteristics of the specific subject under consideration. Thesefactors and their relationship to determining this amount are well knownto skilled practitioners in the medical, veterinary, and other relatedarts. This amount and the method of administration can be tailored toachieve optimal efficacy but will depend on such factors as weight,diet, concurrent medication and other factors which those skilled in theart will recognize.

In one embodiment, the agent is administered for a “therapeuticallyeffective time” refers to the period of time during which apharmaceutically effective amount of a compound is administered, andthat is sufficient to reduce one or more symptoms associated withSARS-coronavirus infection.

The agent may be administered before, concomitantly with, and/or afterdetection of symptoms of infection with SARS-coronavirus. The term“concomitant” when in reference to the relationship betweenadministration of a compound and disease symptoms means thatadministration occurs at the same time as, or during, manifestation ofsymptom associated with SARS-coronavirus infection. Also, theinvention's agents may be administered before, concomitantly with,and/or after administration of another type of drug or therapeuticprocedure.

As used herein, the actual amount encompassed by the term “therapeuticamount” will depend on the nature of agent, route of administration, thetype of subject being treated, and the physical characteristics of thespecific subject under consideration. These factors and theirrelationship to determining this amount are well known to skilledpractitioners in the medical, veterinary, and other related arts. Thisamount and the method of administration can be tailored to achieveoptimal efficacy but will depend on such factors as weight, diet,concurrent medication and other factors which those skilled in the artwill recognize. The dosage amount and frequency are selected to createan effective level of the compound without substantially harmfuleffects. The agent may be administered by, for example, oral, parenteral(e.g., subcutaneous, intravenous, intramuscular, intrastemal injection,and infusion), intranasal, and/or inhalation routes. A therapeuticamount of the agent may be determined using in vitro and in vivo assaysknown in the art.

The agents may be administered with one or more pharmaceuticallyacceptable carrier, diluent or excipient. Pharmaceutically acceptablecarriers are known in the art such as those described in, for example,Remingtons Pharmaceutical Sciences, Mack Publishing Co. (A. R. Gennaroedit. 1985). The pharmaceutically acceptable carriers may be liquid,with the compositions being, for example, an oral syrup or injectableliquid. Compositions in solid or liquid form may include an agent whichbinds to the active component(s) and thereby assists in the delivery ofthe active components. Suitable agents which may act in this capacityinclude a monoclonal or polyclonal antibody, a protein or a liposome.Alternatively, the pharmaceutical composition of the present inventionmay consist of gaseous dosage units, e.g., it may be in the form of anaerosol useful in, for example, inhalatory administration. The term“aerosol” is used to denote a variety of systems ranging from those ofcolloidal nature to systems consisting of pressurized packages. Deliverymay be by a liquefied or compressed gas or by a suitable pump systemwhich dispenses the active ingredients. Aerosols of compounds of theinvention may be delivered in single phase, bi-phasic, or tri-phasicsystems in order to deliver the active ingredient(s). Delivery of theaerosol includes the necessary container, activators, valves,subcontainers, spacers and the like, which together may form a kit.Preferred aerosols may be determined by one skilled in the art, withoutundue experimentation.

Liquid pharmaceutical compositions of the invention, whether they besolutions, suspensions or other like form, may include one or more ofthe following adjuvants: sterile diluents such as water for injection,saline solution, preferably physiological saline, Ringer's solution,isotonic sodium chloride, fixed oils such as synthetic mono ordigylcerides which may serve as the solvent or suspending medium,polyethylene glycols, glycerin, cyclodextrin, propylene glycol or othersolvents; antibacterial agents such as benzyl alcohol or methyl paraben;antioxidants such as ascorbic acid or sodium bisulfite; chelating agentssuch as ethylenediaminetetraacetic acid; buffers such as acetates,citrates or phosphates and agents for the adjustment of tonicity such assodium chloride or dextrose. The parenteral preparation can be enclosedin ampoules, disposable syringes or multiple dose vials made of glass orplastic. Physiological saline is a preferred adjuvant. An injectablepharmaceutical composition is preferably sterile.

J. Producing SARS-CoV and SARS-CoV Polypeptides

The invention also provides a method for producing one or more ofSARS-coronavirus particles and SARS-coronavirus polypeptide, comprising:a) providing: (i) SARS-coronavirus; and (ii) a cell type chosen from oneor more of HEK-293T, Huh-7, Mv1Lu, pRHMK and pCMK; and b) inoculatingthe cell type with the virus under conditions such that the inoculatedcell produces one or more of SARS-coronavirus and SARS-coronaviruspolypeptide. One advantage in using a combination of cells that areinfected with SARS-CoV to generate antibodies and/or vaccines is thateach cell in the combination may differently process the viral proteins.Thus, a combination of cells infected with SARS-CoV would enable thegeneration of antibodies and/or vaccines that are specific to differentviral proteins, thereby increasing the sensitivity and/or specificity ofthe antibodies and/or vaccines in SARS-CoV detection and/or treatment.

In one embodiment, the invention's methods may be used to produce one ormore SARS-coronavirus antigens. The terms “antigen,” “immunogen,”“antigenic,” “immunogenic,” “antigenically active,” and “immunologicallyactive” refer to any molecule that is capable of inducing a specifichumoral or cell-mediated immune response. An immunogen generallycontains at least one epitope. Immunogens are exemplified by, but notrestricted to molecules which contain a peptide, polysaccharide, nucleicacid sequence, and/or lipid. Complexes of peptides with lipids,polysaccharides, or with nucleic acid sequences are also contemplated,including (without limitation) glycopeptide, lipopeptide, glycolipid,etc. These complexes are particularly useful immunogens where smallermolecules with few epitopes do not stimulate a satisfactory immuneresponse by themselves.

The terms “epitope” and “antigenic determinant” refer to a structure onan antigen which interacts with the binding site of an antibody and/or Tcell receptor as a result of molecular complementarity. An epitope maycompete with the intact antigen, from which it is derived, for bindingto an antibody. Generally, secreted antibodies and their correspondingmembrane-bound forms are capable of recognizing a wide variety ofmolecules as antigens, whereas T cell receptors are capable ofrecognizing only fragments of proteins which are complexed with MHCmolecules on cell surfaces. Antigens recognized by immunoglobulinreceptors on B cells are subdivided into three categories: T-celldependent antigens, type 1 T cell-independent antigens; and type 2 Tcell-independent antigens. Also, for example, when a protein or fragmentof a protein is used to immunize a host animal, numerous regions of theprotein may induce the production of antibodies which bind specificallyto a given region or three-dimensional structure on the protein; theseregions or structures are referred to as antigenic determinants. Anantigenic determinant may compete with the intact antigen (i.e., theimmunogen used to elicit the immune response) for binding to anantibody.

Exemplary SARS-coronavirus antigens include, without limitation, atleast a portion of a SARS-CoV polypeptide chosen from one or more ofNucleocapsid (N), Spike glycoprotein (S), Matrix (M), E protein, andReplicase proteins (Pol 1a/b) described supra.

SARS-CoV polypeptides and antigens may be made using methods known inthe art. In one embodiment, SARS-CoV antigens may be obtained bypurifying them using routine methods, from cells (such as the exemplaryHEK-293T, Huh-7, Mv1Lu, pRHMK and/or pCMK cell, etc.) that are infectedwith SARS-CoV.

In another embodiment, SARS-CoV polypeptides (such as antigens) may besynthesized by chemical synthesis. Synthetic chemistry techniques, suchas solid phase Merrifield synthesis are advantageous for reasons ofpurity, freedom from undesired side products, ease of production, etc. Asummary of the techniques available are found in several articles,including Steward et al., Solid Phase Peptide Synthesis, W. H. Freeman,Co., San Francisco (1969); Bodanszky, et al., Peptide Synthesis, JohnWiley and Sons, Second Edition (1976); J. Meienhofer, Hormonal Proteinsand Peptides, 2:46, Academic Press (1983); Merrifield, Adv. Enzymol.32:221-96 (1969); Fields, et al., Intl. Peptide Protein Res., 35:161-214(1990), and U.S. Pat. No. 4,244,946 for solid phase peptide synthesis;and Schroder et al., The Peptides, Vol 1, Academic Press (New York)(1965) for classical solution synthesis. Protecting groups usable insynthesis are described as well in Protective Groups in OrganicChemistry, Plenum Press, New York (1973). Solid phase synthesis methodsconsist of the sequential addition of one or more amino acid residues orsuitably protected amino acid residues to a growing peptide chain.Either the amino or carboxyl group of the first amino acid residue isprotected by a suitable selectively removable protecting group. Adifferent, selectively removable protecting group is utilized for aminoacids containing a reactive side group such as lysine.

In a further embodiment, SARS-CoV polypeptides (such as antigens) may beproduced by expression of recombinant DNA constructs prepared inaccordance with well-known methods. Such production can be desirable toprovide large quantities or alternative embodiments of such compounds.In one embodiment, DNA sequences of open reading frames (ORFs) encodingthe desired peptide sequence is prepared using commercially availablenucleic acid synthesis methods. The chemically synthesized DNA isisolated in a purified form, and inserted into an expression vector, asexemplified by, but not limited to, plasmid, phagemid, shuttle vector,cosmid, and virus.

Expression can be effected in procaryotic, eukaryotic and/or viralhosts. Prokaryotes most frequently are represented by various strains ofE. coli. However, other microbial strains may also be used, such asbacilli, for example Bacillus subtilis, various species of Pseudomonas,or other bacterial strains. In such procaryotic systems, plasmid vectorswhich contain replication sites and control sequences derived from aspecies compatible with the host are used. For example, a workhorsevector for E. coli is pBR322 and its derivatives. Commonly usedprocaryotic control sequences, which contain promoters for transcriptioninitiation, optionally with an operator, along with ribosomebinding-site sequences, include such commonly used promoters as thebeta-lactamase (penicillinase) and lactose (lac) promoter systems, thetryptophan (trp) promoter system, and the lambda-derived PL promoter andN-gene ribosome binding site. However, any available promoter systemcompatible with procaryote expression can be used.

Expression in eukaryotic cells (such as yeast, insect, and mammaliancells) is expressly contemplated. This method is particularly suited forglycoproteins, such as S.

Expression by viral vectors may be achieved using, for example, vacciniaviruses, retroviral vectors, alpha viruses, influenza virus,adenoviruses, and baculoviruses, which may be engineered to express theSARS-CoV polypeptides and/or antigens upon infection/transduction ofvarious cell types. These systems can be used to infect a variety ofcell types from mammalian to insect cells. This approach may be veryefficient resulting in very high level protein expression.

In one embodiment, the SARS-CoV polypeptide (such as antigen) may beisolated following recombinant expression. The terms “isolated,” “toisolate,” “isolation,” “purified,” “to purify,” “purification,” andgrammatical equivalents thereof as used herein, refer to the reductionin the amount of at least one contaminant (such as protein and/ornucleic acid sequence) from a sample. Thus purification results in an“enrichment,” i.e., an increase in the amount of a desirable proteinand/or nucleic acid sequence in the sample. For example, the SARS-CoVpolypeptide (such as antigen) may be fused to another molecule capableof binding to a ligand. The ligand may be immobilized to a solid supportto facilitate isolation of the fused polypeptide. Ligand-binding systemsuseful for the isolation of polypeptides are commercially available andinclude, for example, the staphylococcal protein A and its derivative ZZ(which binds to human polyclonal IgG), histidine tails (which bind toNi²⁺), biotin (which binds to streptavidin), maltose-binding protein(MBP) (which binds to amylose), glutathione S-transferase (which bindsto glutathione), etc. It is not intended that the polypeptide probes ofthe present invention be limited to any particular isolation system. Theuse of 6-8 Histidine tags in combination with Ni²⁺ chromatography hasbeen successfully used for the production of N and E proteins of otherCoVs (e.g., MHV).

In one embodiment, the SARS-CoV particles and/or antigens find use inantibody generation. These antibodies may be used in diagnostic assaysfor the detection of SARS-CoV, as described supra. The antibodies mayalso be used in the prophylaxis and/or treatment of SARS-CoV infection.

In another embodiment, the cells and methods of the invention are usefulfor the production of SARS-CoV particles and/or antigens for use invaccine formulations. The term “vaccine” as used herein refers to apreparation of a pathogenic organism (such as virus as exemplified bySARS-CoV and human immunodeficiency virus, bacterium, fungus, protistsuch as the malaria agent Plasmodium, multicellular parasite such asSchistosoma, etc.) and/or an antigen isolated from the organism, whichcan be administered prophylactially to an animal to induce immunity.Vaccines include, but are not limited to, live attenuated vaccines,inactivated vaccines, and subunit vaccines. Methods for making and usingvaccines are known in the art (Murphy and Chanock, “Immunization againstviral diseases” Chapter 16 pp. 435-467, Eds. Knipe and Howley, Publ.Lippincott Williams and Wilkins, Philadelphia, Field's Virology FourthEdition, 2001).

In one embodiment, the vaccine is a live attenuated vaccine. The term“live attenuated vaccine” refers to a strain (preferably an avirulentstrain) of a pathogenic organism that is nonpathogenic and which stillinduces specific immunity against the pathogenic organism. Methods forproducing live attenuated vaccines are know in the art such as thosewhich use vaccinia virus to vaccinate against smallpox. In oneembodiment, passage of the virulent virus in cell culture can be used toproduce a live attenuated vaccine strain. Such vaccines are exemplifiedby those for measles, mumps and rubella. In another embodiment, liveattenuated vaccines may be produced by introducing site specificmutations into virulence genes to produce an attenuated virus strain forvaccine.

The term “inactivated vaccine” refers to a preparation of a killedand/or inactivated pathogenic organism. Methods for making inactivatedvaccines are known in the art such as by chemical inactivation of virusthat has been grown in eggs or in cell culture. Successful inactivatedvaccines have been produced for rabies and influenza. In one embodiment,inactivated SARS-CoV vaccine may be prepared from virus produced by theinvention's cells (such as the exemplary HEK-293T, Huh-7, Mv1Lu, pRHMKand/or pCMK cell, etc.). The virus is isolated from the culture mediumby affinity chromatography using cellufine sulphate (Palache et al., J.Infect. Dis. 176(suppl. 1):S20-S23 (1997)). The intact live virus isinactivated after purification by any one of a number of methods knownin the art, such as formalin and/or propiolactone treatment to producean inactivated viral vaccine. Virus inactivation may be achieved byincubation of the virus suspension in 0.1% formaldehyde for 10 to 14days at 4° C. The inactivated viral preparation is then tested in amodel mammal using standard protocols, before use in human clinicaltrials.

The term “subunit vaccine” refers to an antigenic polypeptide of thepathogenic organism that has been recombinantly expressed in vitro.Methods for making subunit vaccines are known in the art such as for thehepatitis B virus vaccine which was generated using hepatitis B surfaceantigen expressed in yeast cells. A “subunit vaccine” also refers to arecombinant viral or bacterial vectors that expresses genes encoding anantigenic polypeptide of the pathogenic organism. Exemplary recombinantviral vectors include vaccinia virus, adenovirus, paramyxoviruses, avianpoxviruses, yellow fever virus and vesicular stomatitis virus. A“subunit vaccine” further includes DNA sequences, such as a plasmidcontaining the coding sequence for an antigen that is linked to a strongpromoter sequence that is active in mammalian cells. Such plasmids areinoculated directly into the host, the viral gene is expressed in thehost and antibody and cell-mediated immunity can then be induced to therecombinant antigen.

Vaccines and/or antibodies against SARS-CoV may be used for immunizing amammal against SARS-coronavirus, by administering these compositions togenerating an immune response in the mammal against SARS-coronavirus. Inone embodiment, vaccines and/or antibodies are used therapeutically in amammal that is already infected with SARS-coronavirus. In anotherembodiment, vaccines and/or antibodies are used prophylactically in amammal that is not known to be infected with SARS-coronavirus.

K. Compositions and Methods for Using Protease Inhibitors to ReduceInfection With Plus-Strand RNA Viruses

The invention provides compositions and methods for reducing infectionwith plus-strand RNA viruses. In one embodiment, the invention providesa composition comprising (i) cells susceptible to a virus that is not aplus-strand RNA virus, and (ii) protease inhibitor. The terms“positive-strand RNA virus “plus-strand RNA virus,” and “+-strand RNAvirus,” are equivalent terms that refer to a virus whose genome containsa plus-strand RNA.

Without intending to limit the type of the virus, in one embodiment, the“virus that is not a plus-strand RNA virus” contains a genome of singlestranded DNA, double stranded DNA, double stranded RNA, ornegative-strand RNA. Also without limiting the source of the virus, thevirus that is not a plus-strand RNA virus may be an animal virus, plantvirus, and bacteriophage. More particularly, the animal virus that isnot a plus-strand RNA virus is exemplified by, but not limited to,Arenaviridae, Baculoviridae, Birnaviridae, Bunyaviridae, Cardiovirus,Corticoviridae, Cystoviridae, Epstein-Barr virus, Filoviridae,Hepadnviridae, Hepatitis virus, Herpesviridae, Influenza virus,Inoviridae, Iridoviridae, Metapneumovirus, Orthomyxoviridae, Papovaviru,Paramyxoviridae, Parvoviridae, Polydnaviridae, Poxyviridae, Reoviridae,Rhabdoviridae, Semliki Forest virus, Tetraviridae, Toroviridae, Vacciniavirus, Vesicular stomatitis virus.

Cells that are susceptible to viruses are known in the art and areexemplified, but not limited to, cells susceptible to metapneumovirus,cells susceptible to cells susceptible to Arbovirus (such as BHK-21cells), cells susceptible to BK polyomavirus (such as NCI-H292 cells),cells susceptible to BVDV (such as BT, and EBTr cells), cellssusceptible to CMV (such as H&V-Mix, HEL, HEL-299, HFL-Chang, Hs27(HFF), Human Fetal Tonsil, MRC-5, MRHF, Mv1Lu, and WI-38 cells), cellssusceptible to Coxsackie A (such as MRC-5, RD, HeLa, HEp-2, pMK, MDCK,and E-Mix cells), cells susceptible to Echovirus (such as HEL-299,HFL-Chang, and pMK cells), cells susceptible to Encephalitis (such asCV1 cells), cells susceptible to Herpesviruses (such as Human FetalTonsil, L-929, CHO-K1 cells), cells susceptible to HSV (such as A549,BGMK, CV1, Duck Embryo, EBTr, ELVIS-HSV, H&V-Mix, HEL, HEL-299, HeLa,HEp-2, Hs27 (HFF), LLC-MK2, MDCK, MRC-5, MRHF, Mv1Lu, NCI-H292, pAGMK,pCMK, pRK, RD, RK, RK1, R-Mix, Vero, WI 38 cells), cells susceptible toInfluenza (such as A549, Chicken embryo, HEp-2, LLC-MK2, MDCK, MRC-5,pAGMK, pCMK, pRhMK, R-Mix, WI 38, NCI-H292, and Mv1Lu cells), cellssusceptible to Measles (such as A549, Chicken embryo, CV1, HEp-2,LLC-MK2, NCI-H292, pMK, and Vero cells), cells susceptible to Mumps(such as A549, BGMK, CV1, HEp-2, Hs27 (HFF), LLC-MK2, MRC-5, pCMK, pMK,pRK, RK1, Vero, and WI 38 cells), cells susceptible to Myxovirus (suchas LLC-MK2 cells), cells susceptible to Newcastle disease (such asChicken embryo cells), cells susceptible to Panleukopenia (such asCHO-K1 cells), cells susceptible to Parainfluenza (such as A549, BGMK,HEp-2, Hs27 (HFF), L-929, LLC-MK2, MDCK, MRC-5, MRHF, pAGMK, pCMK,pRhMK, R-Mix, Vero, WI 38, BT, EBTr cells), cells susceptible to canineParvovirus (such as CHO-K1 cells), cells susceptible to felinePicornavirus (such as CHO-K1 cells), most fibroblast and heteroploidcell lines, MRC-5, pCMK, WI 38, HeLa, HeLa S-3, BS-C-2, CV1, Vero,LLC-MK2 cells), cells susceptible to Poxvirus (such as LLC-MK2 cells),cells susceptible to Rabies (such as CHO-K1 and L-929 cells), cellssusceptible to Reovirus (such as MDCK, EBTr, CHO-K1, L-929, NCI-H292cells), cells susceptible to Rhinovirus (such as HEL, HEL-299,HFL-Chang, Hs27 (HFF), LLC-MK2, MRC-5, WI 38, NCI-H292 cells), cellssusceptible to Rotavirus (such as A549, CV1, Vero cells), cellssusceptible to RSV (such as A549, BGMK, HeLa, HEp-2, Hs27 (HFF), HumanFetal Tonsil, MDCK, MRC-5, MRHF, NCI-H292, pRhMK, R-Mix, Vero, WI 38cells), cells susceptible to Rubella (such as BHK-21, BS-C-1, HEp-2,LLC-MK2, pMK, RK13, SIRC, Vero cells), cells susceptible to SV40 (suchas BS-C-3, CV1 cells), cells susceptible to Vaccinia (such as Chickenembryo, EBTr, L-929, NCI-H292 cells), cells susceptible to Vesicularstomatitis (such as BS-C-4, Duck Embryo, HEL-299, HeLa S-5, EBTr cells),and cells susceptible to VZV (such as A549, CV1, H&V-Mix, HEL, HEL-299,HFL-Chang, HNK, Hs27 (HFF), MRC-5, MRHF, SF, Vero, WI 38, M7, pGuineaPig Embryo cells).

Also without intending to limit the source or type of virus, the“plus-strand RNA virus” is exemplified by togavirus, flavivirus,coronavirus, and picornavirus (including Adenovirus, Enterovirus,Immunodeficiency virus, Poliovirus, and Retrovirus).

More particularly, Togaviruses are exemplified by eastern equineencephalitis virus, western equine encephalitis virus, rubella virus. Avariety of infectious agents comprise the alphaviruses (a subgroup oftogaviridae), including Chikungunya, Mayaro, Igbo Ora, Ross River virus,Venezuelan equine encephalitis, Eastern equine encephalitis, and Westernequine encephalitis. While the encephalitides have been discussedpreviously (Small Group 3, Neurotropic Viruses, Nov. 13-15, 2001), thisgroup of “emerging viruses” causes a range of diseases (from acutearthropathy to systemic febrile illness) in various parts of the worldincluding the United States. A wide range of animals host these viruses,including birds, rodents, primates, wallabies, equines, and bats.However, all alphaviruses pathogenic for humans replicate in and aretransmitted by mosquitoes.

Flaviviruses are exemplified by Dengue fever virus, Yellow fever virus,St. Louis encephalitis virus, Japanese B encephalitis virus, West Nilevirus, and Hepatitis C virus.

The term “coronavirus” refers to a virus whose genome is plus-strandedRNA of about 27 kb to about 33 kb in length depending on the particularvirus. The virion RNA has a cap at the 5′ end and a poly A tail at the3′ end. The length of the RNA makes coronaviruses the largest of the RNAvirus genomes. In one embodiment, coronavirus RNAs encode: (1) anRNA-dependent RNA polymerase; (2) N-protein; (3) three envelopeglycoproteins; plus (4) three non-structural proteins. Thesecoronaviruses infect a variety of mammals & birds. They causerespiratory infections (common), enteric infections (mostly ininfants >12 mo.), and possibly neurological syndromes. Coronaviruses aretransmitted by aerosols of respiratory secretions. Coronaviruses areexemplified by, but not limited to, human enteric coV (ATCC accession #VR-1475), human coV 229E (ATCC accession # VR-740), human coV OC43 (ATCCaccession # VR-920), and SARS-coronavirus (Center for Disease Control).

Picornavirus comprises several genuses such as Enterovirus (exemplifiedby human enterovirus A, B, C, and D, porcine enterovirus A and B,Poliovirus, Coxsackie A and B virus, and Echo virus), Rhinovirus(exemplified by Human rhinovirus), Hepatovirus (exemplified by HepatitisA virus), Cardiovirus (exemplified by Encephalomyocarditis virus),Aphthovirus (exemplified by Foot-and-mouth disease virus), Parechovirus(exemplified by Human parechovirus), Erbovirus (exemplified by Equinerhinitis B virus), Kobuvirus (exemplified by Aichi virus), Hepatovirus,and Teschovirus (exemplified by Porcine teschovirus).

Cells susceptible to plus-sense RNA viruses are exemplified by, but notlimited to, cells susceptible to Adenovirus (such as 293, A549, HEL,HEL-299, HEp-2, HFL-Chang, HNK, Hs27, KB, LC-MK2, MDCK, MRC-5, MRHF,NCI-H292, pRK, RK1, R-Mix, Vero, WI 38, HeLa, and HeLa S-4 cells), cellssusceptible to Bovine adenovirus (such as BT cells), cells susceptibleto Bovine enterovirus (such as BT cells), cells susceptible toEnterovirus (such as A549, BGMK, Caco-2, HEL, HEp-2, HNK, Hs27 (HFF),LLC-MK2, MRC-5, MRHF, NCI-H292, pAGMK, pCMK, pRhMK, RD, Vero, and WI 38cells), cells susceptible to feline Calicivirus (such as CHO-K1 cells),cells susceptible to Poliovirus (such as A549, BGMK, FL Amnion, HEL-299,HEp-2, HFL-Chang, Hs27 (HFF), and cells susceptible to bovine Infectiousrhinotracheitis virus (such as BT and EBTr cells).

The invention further provides a method for detecting a virus that isnot a plus-strand RNA virus in a sample, comprising: a) providing: i) asample; ii) cells susceptible to the virus that is not a plus-strand RNAvirus; and iii) one or more protease inhibitor; b) contacting the cellsand the sample in the presence of the protease inhibitor to producecontacted cells, wherein replication of the plus-strand RNA virus in thecontacted cells is not reduced relative to replication of the virus thatis not a plus-strand RNA virus in cells not contacted with the proteaseinhibitor, and wherein replication of a plus-strand RNA virus in thecells contacted with the protease inhibitor is reduced relative toreplication of the plus-strand RNA virus in cells not contacted with theprotease inhibitor.

In one embodiment, the invention provides, compositions and methods forreducing infection with SARS-coronavirus, without substantially reducinginfection with other respiratory viruses. Thus, the invention provides acomposition comprising (i) cells susceptible to a virus chosen frominfluenza virus, parainfluenza virus, adenovirus, metapneumovirus, andrespiratory syncytial virus, and (ii) protease inhibitor. The inventionalso provides a method for detecting a virus chosen from influenzavirus, parainfluenza virus, adenovirus, and respiratory syncytial virusin a sample, comprising: a) providing: i) a sample; ii) cellssusceptible to the virus; and iii) one or more protease inhibitor; b)contacting the cells and the sample in the presence of the proteaseinhibitor to produce contacted cells, wherein infection of the contactedcells by the virus is not reduced relative to cells not contacted withthe protease inhibitor, and wherein infection of the contacted cells bysevere acute respiratory syndrome coronavirus (SARS-coronavirus) isreduced relative to cells not contacted with the protease inhibitor.

These methods are premised, at least in part, on the inventors'discovery that protease inhibitors do not substantially reduce infectionof cells by the exemplary respiratory viruses influenza, parainfluenza,RSV, and adenovirus (Example 8). This is in contrast to the inhibitionin replication of SARS-coronavirus by the cysteine proteinase inhibitor(2S,3S)transepoxysuccinyl-L-leucylamido-3-methylbutane ethyl ester(Yount et al. PNAS 100:12995-13000 (2003).

The invention's methods are useful, where it is desirable to reduceinfectivity by SARS-coronavirus of cells that are routinely used indiagnostic assays of respiratory viruses such as influenza,parainfluenza, RSV, and adenovirus.

As used herein the term “influenza virus” refers to members of theorthomyxoviridae family of enveloped viruses with a segmented antisenseRNA genome (Knipe and Howley (eds.) Fields Virology, 4th edition,Lippincott Williams and Wilkins, Philadelphia, Pa. [2001]). Two types ofinfluenza virus (A and B) are human pathogens causing respiratorypathology. While not intending to limit the type of influenza virus, inone embodiment, the influenza virus is chosen from influenza A,influenza B, and influenza C. Also while not intending to limit the celltype, the cells susceptible to influenza virus comprise cells chosenfrom A549 (Influenza), Chicken embryo (Influenza), HEp-2 (Influenza),LLC-MK2 (Influenza), MDCK (Influenza), MRC-5 (Influenza), pAGMK(Influenza), pCMK (Influenza), pRhMK (Influenza), R-Mix (Influenza), WI38 (Influenza A), NCI-H292 (Influenza A), Mv1Lu (Influenza A,B), andMv1Lu-hF (Influenza A,B). These cells are available from DiagnosticHybrids, Inc., Athens, Ohio.

As used herein, the term “parainfluenza virus” refers to certain membersof the paramyxoviridae genus of enveloped viruses with a single-strandedantisense RNA genome (Knipe and Howley (eds.) Fields Virology, 4thedition, Lippincott Williams and Wilkins, Philadelphia, Pa. [2001]).Four types of parainfluenza virus (1 to 4) are human respiratorypathogens. While not intending to limit the type of parainfluenza virus,in one embodiment, the parainfluenza virus is chosen from parainfluenza1, parainfluenza 2, and parainfluenza 3. In another embodiment, thecells susceptible to parainfluenza virus comprise cells chosen from A549(Parainfluenza), BGMK (Parainfluenza), HEp-2 (Parainfluenza), Hs27 (HFF)(Parainfluenza), L-929 (Parainfluenza), LLC-MK2 (Parainfluenza), MDCK(Parainfluenza), MRC-5 (Parainfluenza), MRHF (Parainfluenza), pAGMK(Parainfluenza), pCMK (Parainfluenza), pRhMK (Parainfluenza), R-Mix(Parainfluenza), Vero (Parainfluenza), WI 38 (Parainfluenza), BT(Parainfluenza 3), and EBTr (Parainfluenza 3). These cells are availablefrom Diagnostic Hybrids, Inc., Athens, Ohio.

As used herein, the term “adenovirus” refers to a double-stranded DNAadenovirus of animal origin, such as avian, bovine, ovine, murine,porcine, canine, simian, and human origin. Avian adenoviruses areexemplified by serotypes 1 to 10 which are available from the ATCC, suchas, for example, the Phelps (ATCC VR-432), Fontes (ATCC VR-280), P7-A(ATCC VR-827), IBH-2A (ATCC VR-828), J2-A (ATCC VR-829), T8-A (ATCCVR-830), and K-1¦ (ATCC VR-921) strains, or else the strains designatedas ATCC VR-831 to 835. Bovine adenoviruses are illustrated by thoseavailable from the ATCC (types 1 to 8) under reference numbers ATCCVR-313, 314, 639-642, 768 and 769. Ovine adenoviruses include the type 5(ATCC VR-1343) or type 6 (ATCC VR-1340). Murine adenoviruses areexemplified by FL (ATCC VR-550) and E20308 (ATCC VR-528). Porcineadenovirus (5359) may also be used. Adenoviruses of canine origininclude all the strains of the CAVI and CAV2 adenoviruses [for example,Manhattan strain or A26/61 (ATCC VR-800) strain]. Simian adenovirusesare also contemplated, and they include the adenoviruses with the ATCCreference numbers VR-591-594, 941-943, and 195-203. Human adenoviruses,of which there greater than fifty (50) serotypes are known in the art,are also contemplated, including the Ad2, Ad3, Ad4, Ad5, Ad7, Ad9, Ad12,Ad17, and Ad40 adenoviruses.

Without limiting the type of cell, the cells susceptible to adenoviruscomprise cells chosen from 293 (Adenovirus), A549 (Adenovirus), HEL(Adenovirus), HEL-299 (Adenovirus), HEp-2 (Adenovirus), HFL-Chang(Adenovirus), HNK (Adenovirus), Hs27 (HFF) (Adenovirus), KB(Adenovirus), LLC-MK2 (Adenovirus), MDCK (Adenovirus), MRC-5(Adenovirus), MRHF (Adenovirus), NCI-H292 (Adenovirus), pRK(Adenovirus), RK1 (Adenovirus), R-Mix (Adenovirus), Vero (Adenovirus),WI 38 (Adenovirus), HeLa (Adenovirus 3), and HeLa S-4 (Adenovirus 5).These cells are available from Diagnostic Hybrids, Inc., Athens, Ohio.

In another embodiment, the cells susceptible to respiratory syncytialvirus comprise cells chosen from A549, BGMK, HeLa, HEp-2, Hs27 (HFF),Human Fetal Tonsil, MDCK, MRC-5, MRHF, NCI-H292, pRhMK, R-Mix, Vero, andWI 38. These cells are available from Diagnostic Hybrids, Inc., Athens,Ohio.

In one embodiment, the goal of reducing infection of cells bySARS-coronavirus while not substantially reducing susceptibility of thecells to influenza, parainfluenza, RSV, and/or adenovirus viruses may beattained by contacting the cells and/or sample that is being tested witha “protease inhibitor,” i.e., an agent that reduces the activity of anenzyme that degrades proteins by hydrolysing peptide bonds between aminoacid residues. Exemplary protease inhibitors include, but are notlimited to, those obtained from Sigma, and listed in catalog 2000-20001,page 845, including AMASTATIN (page 1046), (2S,3R)-3-Amino-2-hydroxy-4-(4-nitrophenyl)-butanoyl-L-leucine(NITROBESTATIN) (page 1046), 4-Amidinophenylmethanesulfonyl Fluoride(AMPSF) (page 84), Antipain (page 1046), α₁-Antitrypsin (page 125),Aprotinin (page 128), BESTATIN (page 160), CHYMOSTATIN (page 1046),CYSTATIN (page 299), 3,4-Dichlorolsocoumarin (page 336), EBELACTONE A(page 382), EBELACTONE B (page 382), ELASTATINAL (page 1047),trans-Epoxysuccinyl-L-leucylamido-(4-guanidino)butane (E-64) (page 393),ethylene diamine tetra-acetic acid (EDTA) (page 1768), EGTA (page 408),Leupeptin (page 1047), α₂-Macroglobulin (page 626), Nle-Sta-Ala-Sta(page 1047), Pepstatin A (page 1048), phenylmethylsulfonylfluoride(PMSF) (page 772), N-(α-Rhamnopyranosyloxy)hydroxy-phosphinyl-Leu-Trp(PHOSPHORAMIDON) (page 1048), TLCK (page 964), TPCK (page 964), TrypsinInhibitor (Soybean) (page 1735), Trypsin Inhibitor (Egg) (page 992),Actinonin (page 128), and Glycyrrhizio Acid (page 489).

In one embodiment the protease inhibitor is a drug that has beenapproved by the FDA. These are exemplified by protease inhibitorsapproved for reducing infection with human immunodeficiency virus (HIV),such as, without limitation AGENERASE (AMPRENAVIR), CRIXIVAN(INDINAVIR), FORTOVASE (SAQUINAVIR), INVIRASE (SAQUINAVIR), KALETRA(LOPINAVIR), LEXIVA (FOSAMPRENAVIR) which is formerly know as GW-433908and VX-175 and is an improved formulation of AGENERASE (AMPRENAVIR),NORVIR (RITONAVIR), REYATAZ (ATAZANAVIR; BMS-232632), and VIRACEPT(NELFINAVIR).

In one embodiment, the cells used in the invention's methods maycomprise a transgenic cell, such as Mv1Lu-hF. In a further embodiment,the contacting further comprises contacting the cells with antibodyspecific for one or more SARS-coronavirus antigen.

The cells may be in single cell type culture or in mixed cell typeculture with a second cell type. The second cell type may comprise awild type cell and/or a transgenic cell. In one embodiment, the mixedcell types comprise mink lung cells such as Mv1Lu cells, and the secondcell type comprises A549 cells (R-mix). In a further embodiment, thecells are frozen in situ, regardless of whether they are in single cellculture or in mixed cell culture.

Where mink lung cells such as Mv1Lu cells are used, the inoculated cellsmay be incubated with the sample for up to 24 hours to reduce the chanceof detecting SARS, while maximizing detection of influenza virus and/orparainfluenza. Data herein (FIG. 4B) shows that SARS was not produced byMv1Lu cells within 24 hours p.i.

The samples that may be used in the invention's methods may be isolatedfrom a mammal such as human, non-human primate, canine, feline, porcine,murine, bovine, avian, hamster, mink.

EXPERIMENTAL

The following examples serve to illustrate certain preferred embodimentsand aspects of the present invention and are not to be construed aslimiting the scope thereof.

Example 1 Materials and Methods

The following is a brief description of the exemplary materials andmethods used in the subsequent Examples.

A. Virus

A seed stock of SARS-CoV Urbani that was passaged twice in Vero E6 cellsprovided by the Centers for Disease Control and Prevention, Atlanta, Ga.This virus was amplified by two passages in Vero E6 cells to establish ahigh titer stock (passage 4) that was utilized for all experiments.SARS-CoV was titered in Vero E6 cells by TCID₅₀. Briefly, cells wereplated in 96-well plates (Falcon, Becton Dickson) at a density of 4×10⁵cells/well in 150 μl of medium. Virus was serially diluted by half logsfrom 10⁰-10⁻⁷ in culture medium containing 2% antibiotic-antimycotic(Invitrogen Corporation, Carlsbad, Calif.). 100 μl of each dilution wasadded per well and cells were incubated 3-4 days at 37° C.

B. Cell Lines

The following Table lists exemplary cell lines that were used and/orequivalent cells that may be used in the invention's methods, and thatare publically available (e.g., from the American Type CultureCollection (ATCC), Rockville, Md., and Diagnostic Hybrids, Inc. (DHI),Athens, Ohio; Cell Bank, Ministry of Health and Welfare, Japan): TABLE 3Exemplary Cells Useful In The Invention Cells Source Vero E6 ATCC #CRL-1586 DHI # 67-0102 MRC-5 ATCC # CCL-171 DHI # 51-0102 BHK-21 ATCC #CCL-10 DHI # 89-0102 MDCK ATCC # CCL-34 DHI # 83-0102 HRT-18 (HCT-18)ATCC # CCL-244 Mv1Lu ATCC # CCL-64 DHI # 58-0102 CMT-93 ATCC # CCL-223AK-D ATCC # CCL-150 A549 ATCC # CCL-185 DHI # 56-0102 HEL DHI # 88-0102pRHMK DHI # 49-T025 DHI # 49-0102 pCMK DHI # 47-T025 DHI # 47-0102 L2ATCC # CCL-149 R-Mix DHI # 96-T025 HEK-293T ATCC # CRL-1573; CRL-11264,CRL-11270; Pear, et al., PNAS USA, Vol 90, pp 8392-8396 September 1993;DuBridge et. al., Mol. Cell. Biol. Vol 7, pp 379-387, 1987; UniversityDr. Yoshi Kawaoka, Univ. Wisconsin, Madison. Huh-7 (JTC-39) CellBank#JCRB0403R-Mix (R-Mix FreshCells™, Diagnostic Hybrids, Inc., Ohio) is a mixedmonolayer of mink lung cells (strain Mv1Lu) and human Adenocarcinomacells (strain A549). the hAPN expression construct used to createBHK21/hAPN and CMT-93/hAPN was previously described (Wentworth, et al.,2001). Further description of Huh-7 cells is in Nakabayashi et al.,Cancer Res., 42: 3858-3863, 1982; Nakabayashi et al., Gann, 75: 151-158,1984; and Nakabayashi et al., Cancer Res., 45:6379-6383, 1985.

Vero E6, 293T, L2, AK-D, A549, pCMK, pRhMK, Mv1Lu, CMT-93, and R-mixwere maintained in Dulbecco's modified Eagle Medium (DMEM) (InvitrogenCorp.) supplemented with 10% fetal bovine serum (FBS) (Hyclone, Logan,Utah) and 2% antibiotic-antimycotic. MDCK cells were maintained in DMEMhigh glucose (Invitrogen Corp.) supplemented with 5% FBS and 2%antibiotic-antimycotic. HEL cells were maintained in Modified Eagle'sMedium (MEM) supplemented with 10% FBS and 2% antibiotic-antimycotic.HRT-18 cells were maintained in RPMI 1640 (Invitrogen Corp.)supplemented with 10% horse serum (Hyclone), 1 mM MEM sodium pyruvate(Invitrogen Corp.) and 2% antibiotic-antimycotic. Huh-7 cells weremaintained in DMEM supplemented with 20% FBS and 2%antibiotic-antimycotic. MRC-5 cells were maintained in MEM supplementedwith 10% FBS, 1 mM sodium pyruvate, 0.1 mM MEM nonessential amino acids(Invitrogen Corp.) and 2% antibiotic-antimycotic. BHK-21 cells weremaintained in DMEM supplemented with 10% FBS and 5% tris phosphatebuffer (Invitrogen Corp.).

C. PCR Assay

G3PDH, genomic SARS-CoV RNA (gRNA) and subgenomic RNA (sgRNA) weredetected using multiplex one-step RT-PCR. Oligonucleotide primers usedto amplify the different targets were as follows: G3P-279 (sense) 5′CATCACCATCTTCCAGGAGC-3′ (SEQ ID NO:72) binds at nt 279-299; G3P-1069R(antisense) 5′-CTTACTCCTTGGAGGCCATG-3′ (SEQ ID NO:73) binds at nt1069-1049; SARS-21,263 (sense) 5′-TGCTAACTACATTTTCTGGAGG-3′ (SEQ IDNO:74) binds at nt 21,263-21,284 of SARS-Urbani; SARS-21,593R(antisense) 5′-AGTATGTTGAGTGTAATTAGGAG-3′ (SEQ ID NO:75) binds at nt21,593-21,571 of SARS-Urbani; and SARS-1 (sense)5′-ATATTAGGTTTTTACCTACCCAGG-3′ (SEQ ID NO:76) binds at nt 1-24 ofSARS-Urbani. Amplification was carried out using the Qiagen® OneStepRT-PCR kit (Qiagen) according to the manufacturer's protocol. Briefly,each reaction consisted of 2 μg of total RNA isolated using TRIZOL®Reagent (Invitrogen), 400 μM dNTPs, 200 nM of each G3PDH primer, 400 nMSARS-1, 400 nM SARS-21,263, 600 nM SARS-21,593R and 2 μl Qiagen enzymemix. The cycling parameters were: 50° C. for 30 min, 95° C. for 15 min,35 cycles of 94° C. for 30 s, 57-58° C. for 30 s, 72° C. for 1 min,followed by 10 min at 72° C. in an Eppendorf Mastercycler gradient(eppendorf). Amplification products were analyzed by electrophoresisthrough a 1.5% agarose gel and visualized by ethidium bromide staining.All primers were synthesized by the Molecular Genetics Core (DavidAxelrod Institute, Wadsworth Center, Albany, N.Y.).

D. Cell Infection

Cells seeded at a density of 2×10⁶ in T25 flasks (Falcon, BectonDickson) were inoculated with virus at an MOI of 0.001 in a final volumeof 1 ml and were incubated 1 h at 37° C. Virus was removed and 5 mlfresh medium added to each flask. Cells were maintained at 37° C.throughout the experiment. At 1, 24 and 48 h post-inoculation (p.i.),cells were observed for CPE, supernatants were collected for subsequenttitration and total RNA was extracted using TRIZOL® Reagent (InvitrogenCorp.). RNA was quantitated by spectrophotometer (Eppendorf).

Example 2 Exemplary Multiplex RT-PCR Assay for the Detection of SARS-CoVReplication

A RT-PCR assay for the detection of SARS-CoV replication was developed.Replication of corona- and arteri-virus RNA occurs through discontinuoussynthesis, thought to occur during negative strand synthesis, generating3′ co-terminal nested subgenomic RNAs (sgRNA). The inventors identifiedtargets within the genome for amplification. Oligonucleotide RT-PCRprimers were designed that amplify genomic SARS-CoV RNA (gRNA) or thesgRNA that is specific to the leader-body junction. Because genomic RNAis present in input virus, the inventors probed for sgRNA, which isindicative of virus entry and/or replication initiation. Genomic RNA wasdetected by amplifying a region between the 1b coding region of thepolymerase gene and the sequence encoding the Spike (S) glycoprotein.Subgenomic RNA was detected using a primer specific to the leadersequence in conjunction with the reverse primer in S that was used forthe gRNA detection. G3PDH primers, designed to amplify G3PDH frommultiple species, served as a positive control for RNA integrity andcDNA production.

To evaluate the RT-PCR assay, Vero E6 cells were inoculated with serialdilutions of SARS-CoV ranging from an MOI of 10⁰ to 10⁻⁸ TCID₅₀/cell.Total RNA was extracted at 1 and 24 h post-inoculation (p.i.). At 1 hp.i. gRNA was detected in cells inoculated with virus at an MOI of 10⁰to 10⁻², as indicated by a band at 300 bp (FIG. 1). Subgenomic RNA wasnot detected (180 bp). However, at 24 h p.i. both gRNA and sgRNA, 300 bpand 180 bp respectively, were detected in cells inoculated with an MOIof 10⁰ to 10⁻⁵. The sgRNA amplicon was confirmed to correspond to the Sleader-body junction sgRNA by sequence analysis (Thiel, et al., 2003, J.Gen. Virol. 84:2305-2315). Genomic RNA was visible at 24 h p.i. in cellsinoculated with an MOI of 10⁻⁷, however this was not seen in repeatedexperiments. The decrease in amplified G3PDH (˜800 bp) as seen in lanes1-6 at 24 h p.i. was consistent between repeated experiments. Thedecrease in G3PDH may be a result of the RT-PCR conditions, which wereoptimized to favor amplification of SARS-CoV gRNA and sgRNA. Individualamplicon were amplified by PCR of cDNAs from the same samples and G3PDHwas consistently detected. Additionally, the decrease in G3PDH may bedue to cell death, which is seen in Vero E6 cells. G3PDH was included asa control for template concentration and RNA integrity, and was alwaysdetected in the absence of viral RNA.

This data demonstrates that the exemplary multiplex RT-PCR assays issensitive for detection of SARS-CoV infection.

Example 3 Primary Monkey Kidney Cells pRhMK and pCMK are Susceptible andPermissive to SARS-CoV

To test the specificity of the RT-PCR assay and to identify cellssusceptible to SARS-CoV, kidney cells derived from two species of monkeywere inoculated with SARS-CoV at an MOI of approximately 0.001. Vero E6cells were included in all experiments as a positive control. Entry andearly replication of SARS-CoV was detected in primary Rhesus monkeykidney cells (pRhMK) and primary Cynomolgous monkey kidney cells (pCMK)at 24 and 48 h p.i. (FIG. 2A). SARS-CoV genomic RNA was detected at 1 hp.i. and increased by 24 and 48 h p.i. In both cell types, sgRNA, absentfrom input virus (1 h) was detected at 24 and 48 h p.i. Once again,G3PDH amplification decreased as the amplification of viral RNAincreased. Subgenomic RNA was not detected in inoculated baby hamsterkidney cells (BHK-21), included as a negative control. In these cells,gRNA was detected only in the viral inoculum (1 h). Both pRhMK and pCMKsupport productive SARS-CoV infection as demonstrated by virus titration(FIG. 2B). Inoculated cells demonstrated an increase in viral titer by48 h p.i. over input virus (2.5 log and 1.5 log increases respectively).At 48 h p.i. supernatants from pRhMK contained virus titers of 5.6×10⁵TCID₅₀ and supernatants from pCMK contained virus at titers of 7.8×10⁴TCID₅₀. The rise in titer was different between the two cell types; pCMKdemonstrated a slower rise in titer from 1 to 24 h p.i. than both pRhMKand Vero E6. Supernatants collected from Vero E6 cells at 48 h p.i.contained viral titers of 3.9×10⁷ TCID₅₀. Consistent with reports byKsiazek et al., cytopathic effect (CPE) was observed in Vero E6 cells asearly as 24 h p.i. (Ksiazek, et al., 2003, N. Engl. J. Med.348:1953-1966). Surprisingly, however, significant CPE was not observedin pRhMK or pCMK 5 days p.i.

This data suggest that CPE may not be an accurate indicator of SARS-CoVreplication. Furthermore, this is the first report of the susceptibilityand permissivity of primary monkey kidney cells pRhMK and pCMK toSARS-CoV.

Example 4 Cells Expressing Known Coronavirus Receptors are notSusceptible to SARS-CoV

Cell lines known to be susceptible to other coronaviruses were assayedfor their susceptibility to SARS-CoV. Human, feline, canine and murinecells expressing known coronavirus receptors were inoculated withSARS-CoV and assayed for viral replication. Cells expressing thereceptor for serogroup 1 coronaviruses (APN) tested included human lungfibroblast-derived cells (MRC-5), canine kidney-derived cells (MDCK),and feline lung epithelia (AK-D). These cells are susceptible to humancoronavirus 229E (HCoV-229E), canine coronavirus (CCoV), and felinecoronavirus (FcoV), respectively. Cells permissive to group 2coronaviruses were also analyzed, including mouse fibroblast derivedcells (L2), that expresses CEACAM 1a, the receptor utilized by MHV-A59and MHV-JHM and a human rectal tumor cell line (HRT-18), known to besusceptible to HCoV-OC43. SARS-CoV gRNA was amplified in all four celllines at 1, 24 and 48 h p.i. (FIG. 3); however, sgRNA was not detectableat any time points post inoculation. A non-specific band (˜220 bp) wasamplified in MRC-5, MDCK and AK-D cells, in all samples including mock.Subgenomic RNA was detected in Vero E6 cells included as a positivecontrol. This data suggest that SARS-CoV utilizes a different receptorthan both group 1 and group 2 coronaviruses.

Example 5 Mv1Lu Cells are Susceptible and Permissive to SARS-CoV

Virology laboratories routinely inoculate cells with clinical specimento identify potential respiratory pathogens. Because little is knownabout the cell types susceptible to SARS-CoV, cells utilized by clinicallaboratories were assayed. R-Mix, a mixed monolayer of mink lung-derivedcells (Mv1Lu) and human lung-derived cells (A549) are used to detect arange of respiratory pathogens. Influenza A and B, adenovirus, RSV andparainfluenza can be detected in Mv1Lu cells while influenza andadenovirus can be detected in A549 cells. Human embryonic lung cells(HEL) are often used to detect rhinovirus and RSV. R-Mix, Mv1Lu, A549and HEL were inoculated with SARS-CoV at an MOI of 0.001. SARS-CoVgenomic RNA was detected in all four cell lines at 1, 24 and 48 h p.i.(FIG. 4A); however, while the gRNA increased from 1 to 48 h p.i. inR-Mix and Mv1Lu cells, it decreased in A549 and HEL cells (FIG. 4).Subgenomic RNA was amplified in R-Mix and Mv1Lu cells at 24 and 48 hp.i. but was not detectable in A549 and HEL cells at any time pointspost inoculation. These results suggest that Mv1Lu cells supportproductive SARS-CoV infection. A non-specific band (˜220 bp) wasamplified in all four cell lines but was present in all samplesincluding the mock infection. Supernatants from R-Mix and Mv1Lu cellswere titered on Vero E6 cells (FIG. 4B). Viral titers decreasedapproximately 0.5 log from 1 h to 24 h p.i. and then increased 1.5 logsby 48 h p.i. Viral titers from Vero E6 cells increased sharply by 4 logsfrom 1 to 24 h p.i. and leveled off. Data herein shows that whileSARS-CoV can productively infect Mv1Lu cells, viral replication occursat much lower levels than that observed in Vero E6 cells.

This is the first report of the susceptibility and permissivity of Mv1Lucells to SARS-CoV.

Example 6 Human Cell Lines HEK-293T and Huh-7 are Susceptible andPermissive to SARS-CoV

Although humans have been infected by SARS-CoV, human-derived cellssusceptible to SARS-CoV infection have not been reported. Humanembryonic kidney-derived cells (HEK-293T) and human liver-derived cells(Huh-7) were inoculated with SARS-CoV at an MOI of 0.001. SARS-CoV gRNAwas detected at 1, 24 and 48 h p.i. in both cell lines, and increasedfrom 1 to 24 h p.i. (FIG. 5A). Subgenomic RNA was amplified at 24 and 48h p.i. in both HEK-293T and Huh-7 cells, indicating that they werepermissive to SARS-CoV infection. MDCK cell, included as a negativecontrol, were negative for sgRNA at all time points. Supernatantscollected at all time points were titered on Vero E6 cells (FIG. 5B). A2-log increase in viral titer (TCID₅₀) was seen at 48 h p.i. in Huh-7cells while an increase of less than 1 log was seen in 293T cells,compared to a 4 log increase in Vero E6 cells. CPE was apparent by 24 hp.i. in Vero E6 cells inoculated at the same time however, no CPE wasobserved in Huh-7 or HEK-293T cells out to 48 h p.i. Surprisingly, theseresults again suggest that CPE is not an accurate indicator of viralreplication in all cell lines.

This is the first report of human cell lines that are susceptible andpermissive to SARS-CoV.

Example 7 Transgenic Cells Expressing Aminopeptidase N are NotPermissive to SARS-CoV

As demonstrated above, MRC-5 cells did not support SARS-CoV RNAreplication suggesting that APN is not sufficient to render cellspermissive to SARS-CoV. However, the human cell lines HEK-293T andHuh-7, shown to be permissive to SARS-CoV replication, express hAPN, thehost cell receptor utilized by HCoV-229E. To further test the role ofAPN in SARS-CoV entry, cells expressing relatively high levels of hAPNon their surface were tested for susceptibility to infection withSARS-CoV. The murine epithelia-derived cell line (CMT-93) and the babyhamster kidney cell line (BHK-21) were transfected with constructsexpressing hAPN to yield CMT-93/hAPN and BHK-21/hAPN (Wentworth et al.2001. J. Virol. 75:9741-9752). These cells, normally non permissive toHcoV-229E infection and replication, were rendered permissive toinfection by expression of hAPN. SARS-CoV genomic RNA was detected inCMT-93, CMT-93/hAPN, BHK-21 and BHK-21/hAPN cells inoculated withSARS-CoV, at 1 to 24 h p.i. (FIG. 6A). The presence of genomic RNA attime points post inoculation varied between experiments. SARS-CoVsubgenomic RNA was not detected at any time points demonstrating thatall four cell lines were non permissive for SARS-CoV replication. HumanAPN was expressed at high levels on both CMT-93/hAPN and BHK-21/hAPNcells as demonstrated by FACS (FIG. 6B). Additionally, Huh-7 cells,included as a positive control for SARS-CoV replication, also expresshigh levels of hAPN as demonstrated by FACS analysis.

Example 8 Protease Inhibitors do not Reduce Infection of Cells by theRespiratory Viruses Influenza, Parainfluenza, and Adenovirus

This Example describes the effect of exemplary protease inhibitors onthe detection of influenza A & B, RSV, adenovirus, and parainfluenza 1,2, and 3 in R-Mix cells.

A. Materials and Methods

Viruses used were influenza A, influenza B, RSV, adenovirus,parainfluenza 1, 2, and 3, all contained in the respiratory virusproficiency panel (Diagnostic Hybrids, Inc., Athens, Ohio). Proteaseinhibitors tested were Actinonin, a leucine aminopeptidase inhibitor(Sigma), Glycyrrhizin, a biologically active derivative of licorice root(Sigma), and E-64, a cysteine protease inhibitor (Sigma). Cells wereR-Mix (Diagnostic Hybrids, Inc., Athens, Ohio) in 48 well plates. Mediumwas RM03T (Diagnostic Hybrids, Inc., Athens, Ohio). Viral detection wasby monoclonal antibody specific for the viruses tested, using a “D3”antibody kit (Diagnostic Hybrids, Inc., Athens, Ohio).

B. Procedure

All inhibitors were dissolved it RM03T to give final concentrations of:Actinonin, 40, 20, and 10 mcg/ml, Glycycrrhizin, 6.08, 1.216, and 0.152mcg/ml, and E-64, 10, 5, 0.5 mcg/ml. Viral stocks were diluted in RM03T.R-Mix 48 well plates containing the appropriate concentration ofinhibitor and additional no-inhibitor control wells were inoculated withthe seven individual viruses separately. The inhibitor wells were induplicate and the control wells were six replicates for each virus.Following inoculation, all plates were centrifuged at 700 g for one hourat room temperature, then incubated in a humidified, CO₂ incubator at37° C. for 24 hours. The cell monolayers were then fixed and stainedaccording to the D3 detection kit instructions. Infected foci werecounted using fluorescent microscopy. The data is shown in Table 4.TABLE 4 Number of infected foci in R-Mix cells treated with respiratoryvirus and protease inhibitors Inhibitor micrograms/ml No inhibitorActinonin Glycycrrhizin E-64 Control* 40** 20 10 6.08 1.216 0.152 10 50.5 FluA 189 ± 12 118 122 171 163 175 218 176 179 174 FluB 455 ± 22 190260 317 273 402 426 323 328 398 Adeno  863 ± 100 147 383 781 1061 938766 865 868 904 RSV 116 ± 12 41 64 103 53 103 130 103 115 107 Para 1 245± 18 160 191 222 295 233 268 207 243 251 Para 2 243 ± 12 151 196 220 281257 265 205 221 223 Para 3 142 ± 14 75 104 115 138 149 138 107 99 127*mean of 6 samples**Evidence of cell toxicityActinonin - a leucine aminopeptidase inhibitorGlycyrrhizin - biologically active plant derivativeE-64 - cysteine protease inhibitorR-Mix lot 960925

The above results demonstrate that protease inhibitors are notinhibitory to infection by any of the seven exemplary viruses that aredetected by Mv1Lu cells and/or R-Mix cells. This is in contrast to theinventors' data demonstrating inhibition in replication of humancoronavirus 229E by the protease inhibitor E64.

All publications and patents mentioned in the above specification areherein incorporated by reference. Various modifications and variationsof the described methods and system of the invention will be apparent tothose skilled in the art without departing from the scope and spirit ofthe invention. Although the invention has been described in connectionwith specific preferred embodiment, it should be understood that theinvention as claimed should not be unduly limited to such specificembodiment. Indeed, various modifications of the described modes forcarrying out the invention which are obvious to those skilled in the artand in fields related thereto are intended to be within the scope of thefollowing claims.

1. A method for detecting replication of severe acute respiratorysyndrome coronavirus (SARS-coronavirus) in a sample, comprisingdetecting the presence SARS-coronavirus subgenomic RNA in said sample byreverse transcriptase polymerase chain reaction (PCR).
 2. The method ofclaim 1, wherein said subgenomic RNA comprises at least a portion of aleader sequence.
 3. The method of claim 1, further comprising detectingSARS-coronavirus genomic RNA.
 4. The method of claim 1, furthercomprising detecting SARS-coronavirus polypeptide.
 5. The method ofclaim 1, further comprising detecting SARS-coronavirus particles.
 6. Amethod for detecting the presence of severe acute respiratory syndromecoronavirus (SARS-coronavirus) in a sample, comprising: a) providing:(i) a sample; and (ii) cells chosen from HEK-293T, Huh-7, Mv1Lu, andpRHMK; b) inoculating said cells with said sample to produce inoculatedcells; and c) detecting the presence of said SARS-coronavirus in saidinoculated cells.
 7. The method of claim 7, wherein said detectingcomprises detecting the presence of subgenomic RNA.
 8. The method ofclaim 6, wherein said detecting comprises detecting the presence ofSARS-coronavirus genomic RNA.
 9. The method of claim 6, wherein saiddetecting comprises detecting the presence of a SARS-coronaviruspolypeptide.
 10. The method of claim 6, wherein said cells comprise atransgenic cell.
 11. The method of claim 10, wherein said transgeniccell comprises a transgenic mink lung epithelial cell line expressinghuman furin, wherein said cell line has a property chosen from (a)increased sensitivity to at least one virus selected from the groupconsisting of influenza A virus, influenza B virus and parainfluenzavirus 3, as compared to Mv1Lu, and (b) enhanced productivity ofinfectious virions upon inoculation with a virus chosen from influenza Avirus, influenza B virus and parainfluenza virus 3, as compared toMv1Lu.
 12. The method of claim 6, wherein said cells comprise a wildtype cell.
 13. The method of claim 6, wherein said cells are in singlecell type culture.
 14. The method of claim 6, wherein said cells are inmixed cell type culture with a second cell type.
 15. The method of claim6, wherein said cells are frozen in situ
 16. The method of claim 6,wherein said sample is isolated from a mammal.
 17. The method of claim6, wherein said mammal is human.
 18. A method for detecting the presenceof severe acute respiratory syndrome coronavirus (SARS-coronavirus) in afirst sample and in a second sample, comprising: a) providing: (i) afirst sample; (ii) a second sample; b) contacting test cells chosen fromHEK-293T, Huh-7, Mv1Lu, and pRHMK with: (i) said first sample to producea first treated sample; and (ii) said second sample to produce a secondtreated sample; wherein said contacting is such that said test cells areinfected with SARS-coronavirus; c) detecting the presence ofSARS-coronavirus genomic RNA and SARS-coronavirus subgenomic RNA in saidfirst treated sample and said second treated sample, wherein saiddetecting indicates the presence of said SARS-coronavirus in said firsttreated sample and said second treated sample.
 19. The method of claim18, wherein said detecting comprises detecting an absence ofSARS-coronavirus subgenomic RNA in said first treated sample.
 20. Themethod of claim 18, wherein said detecting comprises detecting a reducedlevel of SARS-coronavirus subgenomic RNA in said first treated samplecompared to the level of subgenomic RNA in said second treated sample.21. The method of claim 18, wherein said detecting comprises detecting areduced ratio of SARS-coronavirus subgenomic RNA level toSARS-coronavirus genomic RNA level in said first treated sample comparedto said ratio in said second treated sample.
 22. The method of claim 18,wherein said first sample and said second sample are isolated from amammal.
 23. The method of claim 18, wherein said mammal is human.
 24. Amethod for identifying a test agent as altering replication of severeacute respiratory syndrome coronavirus (SARS-coronavirus) in a cell,comprising: a) providing cells treated with a first test agent, whereinsaid cells are chosen from HEK-293T, Huh-7, Mv1Lu, and pRHMK; and b)detecting an altered level of replication of SARS-coronavirus in cellstreated with said first test agent compared to a level of replication ofSARS-coronavirus in cells not treated with said first test agent,wherein said detecting identifies said first test agent as alteringreplication of SARS-coronavirus in a cell.
 25. The method of claim 24,wherein said altered level is a reduced level.
 26. The method of claim24, wherein said altered level is an increased level.
 27. The method ofclaim 24, wherein said detecting comprises detecting SARS-coronavirussubgenomic RNA.
 28. The method of claim 24, wherein said detectingcomprises detecting SARS-coronavirus genomic RNA.
 29. The method ofclaim 24, wherein said detecting comprises detecting SARS-coronaviruspolypeptide.
 30. The method of claim 24, further comprising detectingSARS-coronavirus particles.
 31. The method of claim 24, wherein saiddetecting comprises detecting an absence of SARS-coronavirus genomic RNAin said cells treated with said first test agent.
 32. The method ofclaim 24, wherein said detecting comprises detecting a reduced level ofSARS-coronavirus subgenomic RNA in said cells treated with said firsttest agent compared to the level of SARS-coronavirus subgenomic RNA insaid cells that are not treated with said first test agent.
 33. Themethod of claim 32, wherein detecting an increased reduction in thelevel of SARS-coronavirus subgenomic RNA in said cells treated with saidfirst test agent compared to said cells treated with said second testagent identifies said first test agent as more efficacious than saidsecond test agent in reducing replication of SARS-coronavirus in a cell.34. The method of claim 24, wherein said detecting comprises detecting areduced ratio of SARS-coronavirus subgenomic RNA level relative toSARS-coronavirus genomic RNA level in said cells treated with said firsttest agent compared to said ratio in said cells that are not treatedwith said first test agent.
 35. The method of claim 34, whereindetecting an increased reduction in said ratio of SARS-coronavirussubgenomic RNA level to SARS-coronavirus genomic RNA level in said cellstreated with said first test agent compared to said ratio in said cellstreated with said second test agent identifies said first test agent asmore efficacious than said second test agent in reducing replication ofSARS-coronavirus in a cell.
 36. An antibody specific for one or moreSARS-coronavirus antigen that is produced by a cell chosen fromHEK-293T, Huh-7, Mv1Lu, and pRHMK.
 37. The antibody of claim 36, whereinsaid antibody comprises a polyclonal antibody.
 38. The antibody of claim36, wherein said antibody comprises a monoclonal antibody.
 39. Theantibody of claim 36, wherein said antibody comprises a humanizedantibody.
 40. A composition comprising (i) cells susceptible to a virusthat is not a plus-strand RNA virus, and (ii) protease inhibitor.
 41. Amethod for detecting a virus that is not a plus-strand RNA virus in asample, comprising: a) providing: i) a sample; ii) cells susceptible tosaid virus that is not a plus-strand RNA virus; and iii) one or moreprotease inhibitor; b) contacting said cells and said sample in thepresence of said protease inhibitor to produce contacted cells, whereinreplication of said virus that is not a plus-strand RNA virus in saidcontacted cells is not reduced relative to replication of said virusthat is not a plus-strand RNA virus in cells not contacted with saidprotease inhibitor, and wherein replication of a plus-strand RNA virusin said cells contacted with said protease inhibitor is reduced relativeto replication of said plus-strand RNA virus in cells not contacted withsaid protease inhibitor.
 42. The method of claim 41, wherein said virusthat is not a plus-strand RNA virus is chosen from influenza virus,parainfluenza virus, adenovirus, respiratory syncytial virus, andmetapneumovirus.
 43. The method of claim 42, wherein said influenzavirus is chosen from influenza A, influenza B, and influenza C.
 44. Themethod of claim 42, wherein said parainfluenza virus is chosen fromparainfluenza 1, parainfluenza 2, parainfluenza 3, and parainfluenza 4.45. The method of claim 42, wherein said adenovirus is chosen fromadenovirus 2, adenovirus 3, adenovirus 4, adenovirus 5, adenovirus 7,adenovirus 9, adenovirus 12, adenovirus 17, and adenovirus
 40. 46. Themethod of claim 41, wherein said plus-strand RNA virus is chosen fromtogavirus, flavivirus, coronavirus, and picornavirus.
 47. The method ofclaim 46, wherein said coronavirus comprises SARS-coronavirus.